Stabilized bioactive peptides and methods of identification, synthesis, and use

ABSTRACT

An intracellular selection system allows screening for peptide bioactivity and stability. Randomized recombinant peptides are screened for bioactivity in a tightly regulated expression system, preferably derived from the wild-type lac operon. Bioactive peptides thus identified are inherently protease- and peptidase-resistant. Also provided are bioactive peptides stabilized by a stabilizing group at the N-terminus, the C-terminus, or both. The stabilizing group can be a small stable protein, such as the Rop protein, glutathione sulfotransferase, thioredoxin, maltose binding protein, or glutathione reductase, an α-helical moiety, or one or more proline residues.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. Ser. No. 11/213,668, filed 26Aug. 2005 issued as U.S. Pat. No. 7,365,162 on Apr. 23, 2008), which isa continuation of U.S. Ser. No. 10/210,023, filed 31 Jul. 2002 (nowabandoned), which is a continuation-in-part of U.S. Ser. No. 09/701,947,filed Dec. 5, 2000 (issued as U.S. Pat. No. 6,818,611 on Nov. 16, 2004),which is a National Stage application under 35 U.S.C. §371PCT/US99/23731, filed Oct. 12, 1999 (published as WO/0022112 on Apr. 20,2000), which in turn claims the benefit of U.S. Provisional PatentApplications Serial Nos. 60/104,013, filed Oct. 13, 1998, and60/112,150, filed Dec. 14, 1998, each of which is incorporated herein byreference in its entirety.

TECHNICAL FIELD

This invention relates to stabilized bioactive peptides, and moreparticularly to bioactive peptides that contain heterologous stabilizinggroups attached to one or both of the bioactive peptide's termini.

BACKGROUND

Bioactive peptides are small peptides that elicit a biological activity.Since the discovery of secretin in 1902, over 500 of these peptideswhich average 20 amino acids in size have been identified andcharacterized. They have been isolated from a variety of systems,exhibit a wide range of actions, and have been utilized as therapeuticagents in the field of medicine and as diagnostic tools in both basicand applied research. Tables 1 and 2 list some of the best knownbioactive peptides.

TABLE 1 Bioactive peptides utilized in medicine Size in Amino NameIsolated From Acids Therapeutic Use Angiotensin II Human Plasma 8Vasoconstrictor Bradykinin Human Plasma 9 Vasodilator Caerulein FromSkin 10 Choleretic Agent Calcitonin Human Parathyroid 32 CalciumRegulator Gland Cholecystokinin Porcine Intestine 33 Choleretic AgentCorticotropin Porcine Pituitary 39 Hormone Gland Eledoisin Octopod Venom11 Hypotensive Agent Gastrin Porcine Stomach 17 Gastric ActivatorGlucagon Porcine Pancreas 29 Antidiabetic Agent Gramicidin D Bacillusbrevis 11 Antibacterial Bacteria Agent Insulin Canine PancreasAntidiabetic Agent Insulin A 21 Insulin B 30 Kallidin Human Plasma 10Vasodilator Luteinizing Bovine Hypothalamus 10 Hormone Hormone-Stimulator Releasing Factor Melittin Bee Venom 26 Antirheumatic AgentOxytocin Bovine Pituitary Gland 9 Oxytocic Agent Secretin CanineIntestine 27 Hormone Sermorelin Human Pancreas 29 Hormone StimulatorSomatostatin Bovine Hypothalamus 14 Hormone Inhibitor Vasopressin BovinePituitary Gland 9 Antidiuretic Agent

TABLE 2 Bioactive peptides utilized in applied research Size in AminoName Isolated From Acids Biological Activity Atrial Natriuretic RatAtria 28 Natriuretic Agent Peptide Bombesin Frog Skin 14 GastricActivator Conantokin G Snail Venom 17 Neurotransmitter Conotoxin G1Snail Venom 13 Neuromuscular Inhibitor Defensin HNP-1 Human Neutrophils30 Antimicrobial Agent Delta Sleep- Rabbit Brain 9 Neurological AffectorInducing Peptide Dermaseptin Frog Skin 34 Antimicrobial Agent DynorphinPorcine Brain 17 Neurotransmitter EETI II Ecballium elaterium 29Protease Inhibitor seeds Endorphin Human Brain 30 NeurotransmitterEnkephalin Human Brain 5 Neurotransmitter Histatin 5 Human Saliva 24Antibacterial Agent Mastoparan Vespid Wasps 14 Mast Cell DegranulatorMagainin 1 Frog Skin 23 Antimicrobial Agent Melanocyte Porcine Pituitary13 Hormone Stimulator Stimulating Gland Hormone Motilin Canine Intestine22 Gastric Activator Neurotensin Bovine Brain 13 NeurotransmitterPhysalaemin Frog Skin 11 Hypotensive Agent Substance P Horse Intestine11 Vasodilator Vasoactive Porcine Intestine 28 Hormone IntestinalPeptide

Where the mode of action of these peptides has been determined, it hasbeen found to be due to the interaction of the bioactive peptide with aspecific protein target. In most of the cases, the bioactive peptideacts by binding to and inactivating its protein target with extremelyhigh specificities. Binding constants of these peptides for theirprotein targets typically have been determined to be in the nanomolar(nM, 10⁻⁹ M) range with binding constants as high as 10⁻¹² M (picomolarrange) having been reported. Table 3 shows target proteins inactivatedby several different bioactive peptides as well as the binding constantsassociated with binding thereto.

TABLE 3 Binding constants of bioactive peptides Size in Amino BindingBioactive Peptide Acids Inhibited Protein Constant α-Conotoxin GIA 15Nicotinic 1.0 × 10⁻⁹M Acetylcholine EETI II 29 Trypsin 1.0 × 10⁻¹²M H2(7-5) 8 HSV Ribonucleotide 3.6 × 10⁻⁵M Reductase Histatin 5 24Bacteroides gingivalis 5.5 × 10⁻⁸M Protease Melittin 26 Calmodulin 3.0 ×10⁻⁹M Myotoxin (29-42) 14 ATPase 1.9 × 10⁻⁵M Neurotensin 13 NiRegulatory Protein 5.6 × 10⁻¹¹M Pituitary Adenylate 38 Calmodulin 1.5 ×10⁻⁸M Cyclase Activating Polypeptide PKI (5-24) 20 CAMP-Dependent 2.3 ×10⁻⁹M Protein Kinase SCP (153-180) 27 Calpain 3.0 × 10⁻⁸M Secretin 27HSR G Protein 3.2 × 10⁻⁹M Vasoactive Intestinal 28 GPRNI G Protein 2.5 ×10⁻⁹M Peptide

Recently, there has been an increasing interest in employingsynthetically derived bioactive peptides as novel pharmaceutical agentsdue to the impressive ability of the naturally occurring peptides tobind to and inhibit specific protein targets. Synthetically derivedpeptides could be useful in the development of new antibacterial,antiviral, and anticancer agents. Examples of synthetically derivedantibacterial or antiviral peptide agents would be those capable ofbinding to and preventing bacterial or viral surface proteins frominteracting with their host cell receptors, or preventing the action ofspecific toxin or protease proteins. Examples of anticancer agents wouldinclude synthetically derived peptides that could bind to and preventthe action of specific oncogenic proteins.

To date, novel bioactive peptides have been engineered through the useof two different in vitro approaches. The first approach producescandidate peptides by chemically synthesizing a randomized library of6-10 amino acid peptides (J. Eichler et al., Med. Res. Rev. 15: 481-496(1995); K. Lam, Anticancer Drug Des. 12:145-167 (1996); M. Lebl et al.,Methods Enzymol. 289:336-392 (1997)). In the second approach, candidatepeptides are synthesized by cloning a randomized oligonucleotide libraryinto a Ff filamentous phage gene, which allows peptides that are muchlarger in size to be expressed on the surface of the bacteriophage (H.Lowman, Ann. Rev. Biophys. Biomol. Struct. 26: 401-424 (1997); G. Smithet al., et al. Meth. Enz. 217: 228-257 (1993)). To date, randomizedpeptide libraries up to 38 amino acids in length have been made, andlonger peptides are likely achievable using this system. The peptidelibraries that are produced using either of these strategies are thentypically mixed with a preselected matrix-bound protein target. Peptidesthat bind are eluted, and their sequences are determined. From thisinformation new peptides are synthesized and their inhibitory propertiesare determined. This is a tedious process that only screens for onebiological activity at a time.

Although these in vitro approaches show promise, the use ofsynthetically derived peptides has not yet become a mainstay in thepharmaceutical industry. The primary obstacle remaining is that ofpeptide instability within the biological system of interest asevidenced by the unwanted degradation of potential peptide drugs byproteases and/or peptidases in the host cells. There are three majorclasses of peptidases which can degrade larger peptides: amino andcarboxy exopeptidases which act at either the amino or the carboxyterminal end of the peptide, respectively, and endopeptidases which acton an internal portion of the peptide. Aminopeptidases,carboxypeptidases, and endopeptidases have been identified in bothprokaryotic and eukaryotic cells. Many of those that have beenextensively characterized were found to function similarly in both celltypes. Interestingly, in both prokaryotic and eukaryotic systems, manymore aminopeptidases than carboxypeptidases have been identified todate.

Approaches used to address the problem of peptide degradation haveincluded the use of D-amino acids or modified amino acids as opposed tothe naturally occurring L-amino acids (e.g., J. Eichler et al., Med ResRev. 15:481 496 (1995); L. Sanders, Eur. J. Drug Metabol.Pharmacokinetics 15:95-102 (1990)), the use of cyclized peptides (e.g.,R. Egleton, et al., Peptides 18:1431-1439 (1997)), and the developmentof enhanced delivery systems that prevent degradation of a peptidebefore it reaches its target in a patient (e.g., L. Wearley, Crit. Rev.Ther. Drug Carrier Syst. 8:331-394 (1991); L. Sanders, Eur. J. DrugMetabol. Pharmacokinetics 15: 95-102 (1990)). Although these approachesfor stabilizing peptides and thereby preventing their unwanteddegradation in the biosystem of choice (e.g., a patient) are promising,there remains no way to routinely and reliably stabilize peptide drugsand drug candidates. Moreover, many of the existing stabilization anddelivery methods cannot be directly utilized in the screening anddevelopment of novel useful bioactive peptides. A biological approachthat would serve as both a method of stabilizing peptides and a methodfor identifying novel bioactive peptides would represent a much neededadvance in the field of peptide drug development.

SUMMARY OF THE INVENTION

The present invention provides an intracellular screening method foridentifying novel bioactive peptides. A host cell is transformed with anexpression vector comprising a tightly regulable control region operablylinked to a nucleic acid sequence encoding a peptide. Typically, theencoded peptide has a stabilizing group positioned at one or both endsof the peptide. The transformed host cell is first grown underconditions that repress expression of the peptide and then,subsequently, expression of the peptide is induced. Phenotypic changesin the host cell upon expression of the peptide are indicative ofbioactivity, and are evaluated. If, for example, expression of thepeptide is accompanied by complete or partial inhibition of host cellgrowth, the expressed peptide constitutes a bioactive peptide, in thatit functions as an inhibitory peptide.

Intracellular identification of bioactive peptides can be advantageouslycarried out in a pathogenic microbial host cell. Bioactive peptideshaving antimicrobial activity are readily identified in a microbial hostcell system. Further, the method can be carried out in a host cell thathas not been modified to reduce or eliminate the expression of naturallyexpressed proteases or peptidases. When carried out in a host cellcomprising proteases and peptides, the selection process of theinvention is biased in favor of bioactive peptides that are protease andpeptidase-resistant.

The tightly regulable control region of the expression vector used totransform the microbial host cell according to the invention can bederived from the wild-type Escherichia coli lac operon, and thetransformed host cell can include an amount of Lac repressor proteineffective to repress expression of the peptide during host cell growthunder repressed conditions. To insure a sufficient amount of Lacrepressor protein, the host cell can be transformed with a second vectorthat overproduces Lac repressor protein.

Optionally, the expression vector used to transform the host cell can begenetically engineered to encode a stabilized peptide that is resistantto peptidases and proteases. For example, the coding sequence can bedesigned to encode a stabilizing group at either or both of thepeptide's N-terminus or C-terminus. As another example, the codingsequence can be designed to encode a stabilizing motif such as anα-helix motif or an opposite charge ending motif, as described below.The presence of a stabilizing group at a peptide terminus and/or of astabilizing motif can slow down the rate of intracellular degradation ofthe peptide.

A plurality of vectors can be used to screen a randomized library ofcandidate bioactive peptides.

The present invention also provides a polypeptide that includes abioactive peptide and a stabilizing group coupled to at least oneterminus of the bioactive peptide. Preferably, the bioactive peptide is50 or fewer amino acids in length. The stabilizing group is heterologousto the bioactive peptide and can be, for example, a proline, aproline-containing peptide, a single α-helix or multiple helix bundle,or other polypeptide or small protein such as Rop, human serum albumin,and the like. In one embodiment of the stabilized polypeptide, thestabilizing group(s) lack the capacity to participate in the formationof an intramolecular disulfide bond within the polypeptide. Thus, thestabilizing group is preferably not a thioredoxin polypeptide.

When a polypeptide includes a stabilizing group on each terminus, thestabilizing groups can be the same or different. If different, thestabilizing groups are optionally heterologous to each other, as thatterm is defined below. The first and second stabilizing groups can, butneed not, interact to form a naturally occurring secondary or tertiarystructure. Further, the first and second stabilizing groups can, butneed not, confine the N-terminus and the C-terminus of the bioactivepeptide in close proximity.

The invention further includes a nucleic acid encoding the polypeptideof the invention. A vector that contains such a nucleic acid is alsoincluded. Preferably the vector contains a tightly regulable expressioncontrol sequence operably linked to the nucleic acid sequence encodingthe stabilized polypeptide.

The present invention also includes a method for making a stabilizedpolypeptide that involves coupling a stabilizing group to at least oneterminus of a bioactive peptide. Coupling can be achieved chemically orenzymatically, or can occur as the result of translation in a host cellof a vector containing a nucleic acid sequence encoding the stabilizedpolypeptide. The vector comprises an expression control sequenceoperably linked to the coding sequence; preferably, the expressioncontrol sequence is tightly regulable in said host cell. Optionally themethod includes determining stability of said stabilized polypeptiderelative to said bioactive peptide.

When the method is performed in a host cell, the host cell is firsttransformed with an exogenous nucleic acid encoding the stabilizedpolypeptide, then the stabilized polypeptide is expressed and recovered.The host cells can be prokaryotic, such as bacteria, or eukaryotic.

Phage display can be used to identify a bioactive peptide that can besubsequently stabilized according to the invention. When displayed onthe surface of a bacteriophage, bioactive peptides are tethered at oneend by a bacteriophage protein. The free bioactive peptide (i.e.,uncoupled from the bacteriophage protein) may exhibit a lack ofstability in vivo. Hence, the invention involves stabilizing thesebioactive peptides by coupling them to a stabilizing group at the endthat had been tethered during phage display, thereby effectivelyreplacing the bacteriophage protein as a stabilizing feature. Couplingcan take place chemically, enzymatically, or by way of recombinantgenetic engineering, as described herein. Polypeptides thus stabilizedare also included in the invention.

Alternatively, the stabilized polypeptide can be produced as a directproduct of phage display. A bacteriophage that contains an exogenousnucleic acid encoding a polypeptide comprising a bioactive peptide (orcandidate bioactive peptide), a bacteriophage protein coupled to oneterminus of the bioactive peptide, and a stabilizing group coupled tothe other terminus of the bioactive peptide is cultured under conditionsto cause the bacteriophage to express the stabilized polypeptide anddisplay it on its surface. The stabilizing group can be coupled toeither end of the bioactive peptide, and the bacteriophage protein iscoupled to the other end. Optionally the stabilized polypeptide iscleaved from the host cell surface to yield a stabilized bioactivepeptide comprising the bioactive peptide and the stabilizing group.

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention pertains. Although methods and materialssimilar or equivalent to those described herein can be used in thepractice or testing of the present invention, suitable methods andmaterials are described below. All publications, patent applications,patents, and other references mentioned herein are incorporated byreference in their entirety. In case of conflict, the presentspecification, including definitions, will control. In addition, thematerials, methods, and examples are illustrative only and not intendedto be limiting.

Other features and advantages of the invention will be apparent from thefollowing detailed description and from the claims.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the control region (SEQ ID NO: 1) of the wild-type lacoperon from the auxiliary operator O3 through the translational start ofthe lacZ gene. DNA binding sites include the operators O3 and O1 (bothunderlined), catabolite gene activator protein (CAP) (boxed), the −35site (boxed), and the −10 site (boxed), while important RNA and proteinsites include the Lac1 translation stop site (TGA), the +1 lacZtranscription start site, the Shine Dalgarno (SD) ribosome binding sitefor lacZ, and the LacZ translation start site (ATG).

FIG. 2 is a map of plasmid pLAC11. The unique restriction sites and thebase pair at which they cut are indicated. Other sites of interest arealso shown, including Tet (98-1288), Rop (1931-2122), origin ofreplication (ori) (2551-3138), Amp (3309-25 4 169), and lacPO(4424-4536).

FIG. 3 is a map of plasmid pLAC22. The unique restriction sites and thebase pair at which they cut are indicated. Other sites of interest arealso shown, including Tet (98-1288), Rop (1927-2118), ori (2547-3134),Amp (3305-4165), lacI^(q) (4452-5536), and lacPO (5529-5641).

FIG. 4 is a map of plasmid pLAC33. The unique restriction sites and thebase pair at which they cut are indicated. Other sites of interest arealso shown, including Tet (98-1288), ori (1746-2333), Amp (2504-3364),and lacPO (3619-3731).

FIG. 5 shows the response of the pLAC11-lacZ construct (open circles) tovarying amounts of isopropyl β-D-thiogalactoside (IPTG). A filled squareindicates the β-galactosidase activity that was obtained when MG1655 orCSH27 cells were grown in rich media induced with 1 mM IPTG, while afilled diamond indicates the β-galactosidase activity that was obtainedwhen MG1655 or CSH27 cells were grown in M9 minimal lactose media.

FIG. 6 shows growth curves depicting the inhibitory effects of a two dayinhibitor (pPep12) versus a one day inhibitor (pPep1). Data points forthe control, pLAC 11, for pPep 1, and for pPep 12, are indicated bysquares, circles, and triangles, respectively.

FIG. 7 is a map of the p-Rop(C) fusion vector. The unique restrictionsites and the base pair at which they cut are indicated. Other sites ofinterest are also shown, including Rop (7-198), ori (627-1214), Amp(2245-1385), lacPO (2500-2612).

FIG. 8 is a map of the p(N)Rop-fusion vector. The unique restrictionsites and the base pair at which they cut are indicated. Other sites ofinterest are also shown: Rop (7-204), ori (266-853), Amp (1024-1884),lacPO (2139-2251).

FIG. 9 illustrates a peptide (SEQ ID NO:2) having the opposite chargeending motif, wherein the amino and carboxy termini of the peptide arestabilized by the interactions of the opposite charge ending aminoacids.

DETAILED DESCRIPTION

The present invention represents a significant advance in the art ofpeptide drug development by allowing concurrent screening for peptidebioactivity and stability. Randomized recombinant peptides are screenedfor bioactivity in a tightly regulated inducible expression system thatpermits essentially complete repression of peptide expression in thehost cell. Subsequent induction of peptide expression can then be usedto identify peptides that inhibit host cell growth or possess otherbioactivities.

Intracellular screening of randomized peptides has many advantages overexisting methods. Bioactivity is readily apparent, many diversebioactivities can be screened for simultaneously, very large numbers ofpeptides can be screened using easily generated peptide libraries, andthe host cell, if desired, can be genetically manipulated to identify anaffected protein target. Advantageously, randomized peptides can bescreened in a host cell that is identical to or closely resembles theeventual target cell for antimicrobial, anticancer, and othertherapeutic applications. An additional and very important feature ofthis system is that selection is naturally biased in favor of peptidesthat are stable in an intracellular environment, i.e., that areresistant to proteases and peptidases. Fortuitously, bacterialpeptidases are very similar to eukaryotic peptidases. Peptides that arestable in a bacterial host are thus likely to be stable in a eukaryoticcell as well, allowing bacterial cells to be used in initial screens toidentify drugs that may eventually prove useful as human or animaltherapeutics.

The invention is directed to the identification and use of bioactivepeptides. A bioactive peptide is a peptide having a biological activity.The term “bioactivity” as used herein includes, but is not limited to,any type of interaction with another biomolecule, such as a protein,glycoprotein, carbohydrate, for example an oligosaccharide orpolysaccharide, nucleotide, polynucleotide, fatty acid, hormone, enzyme,cofactor or the like, whether the interactions involve covalent ornoncovalent binding. Bioactivity further includes interactions of anytype with other cellular components or constituents including salts,ions, metals, nutrients, foreign or exogenous agents present in a cellsuch as viruses, phage and the like, for example binding, sequestrationor transport-related interactions. Bioactivity of a peptide can bedetected, for example, by observing phenotypic effects in a host cell inwhich it is expressed, or by performing an in vitro assay for aparticular bioactivity, such as affinity binding to a target molecule,alteration of an enzymatic activity, or the like. Examples of bioactivepeptides include antimicrobial peptides and peptide drugs. Antimicrobialpeptides are peptides that adversely affect a microbe such as abacterium, virus, protozoan, or the like. Antimicrobial peptidesinclude, for example, inhibitory peptides that slow the growth of amicrobe, microbiocidal peptides that are effective to kill a microbe(e.g., bacteriocidal and virocidal peptide drugs, sterilants, anddisinfectants), and peptides effective to interfere with microbialreproduction, host toxicity, or the like. Peptide drugs for therapeuticuse in humans or other animals include, for example, antimicrobialpeptides that are not prohibitively toxic to the patient, and peptidesdesigned to elicit, speed up, slow down, or prevent various metabolicprocesses in the host such as insulin, oxytocin, calcitonin, gastrin,somatostatin, anticancer peptides, and the like.

The term “peptide” as used herein refers to a plurality of amino acidsjoined together in a linear chain via peptide bonds. Accordingly, theterm “peptide” as used herein includes a dipeptide, tripeptide,oligopeptide and polypeptide. A dipeptide contains two amino acids; atripeptide contains three amino acids; and the term oligopeptide istypically used to describe peptides having between 2 and about 50 ormore amino acids. Peptides larger than about 50 are often referred to aspolypeptides or proteins. For purposes of the present invention, a“peptide” is not limited to any particular number of amino acids.Preferably, however, the peptide contains about 2 to about 50 aminoacids (e.g., about 5 to about 40 amino acids, about 5 to about 20 aminoacids, or about 7 to 15 amino acids).

The library used to transform the host cell is formed by cloning arandomized, peptide-encoding oligonucleotide into a nucleic acidconstruct having a tightly regulable expression control region. Anexpression control region can be readily evaluated to determine whetherit is “tightly regulable,” as the term is used herein, by bioassay in ahost cell engineered to contain a mutant nonfunctional gene “X.”Transforming the engineered host cell with an expression vectorcontaining a tightly regulable expression control region operably linkedto a cloned wild-type gene “X” will preserve the phenotype of theengineered host cell under repressed conditions. Under inducedconditions, however, the expression vector containing the tightlyregulable expression control region that is operably linked to thecloned wild-type gene “X” will complement the mutant nonfunctional geneX to yield the wild-type phenotype. In other words, a host cellcontaining a null mutation which is transformed with a tightly regulableexpression vector capable of expressing the chromosomally inactivatedgene will exhibit the null phenotype under repressed conditions; butwhen expression is induced, the cell will exhibit a phenotypeindistinguishable from the wild-type cell. It should be understood thatthe expression control region in a tightly regulable expression vectorof the present invention can be readily modified to produce higherlevels of an encoded biopeptide, if desired (see, e.g., Example 1,below). Such modification may unavoidably introduce some “leakiness”into expression control, resulting in a low level of peptide expressionunder repressed conditions.

In one embodiment, the expression control region of the inducibleexpression vector is derived from the wild-type E. coli lacpromoter/operator region. The expression vector can contain a regulatoryregion that includes the auxiliary operator O3, the CAP binding region,the −35 promoter site, the −10 promoter site, the operator O1, theShine-Dalgarno sequence for lacZ, and a spacer region between the end ofthe Shine-Dalgarno sequence and the ATG start of the lacZ codingsequence (see FIG. 1).

It is to be understood that variations in the wild-type nucleic acidsequence of the lac promoter/operator region can be tolerated in theexpression control region of the preferred expression vector and areencompassed by the invention, provided that the expression controlregion remains tightly regulable as defined herein. For example, the −10site of the wild-type icac operon (TATGTT) is weak compared to thebacterial consensus −10 site sequence TATAAT, sharing four out of sixpositions. It is contemplated that other comparably weak promoters areequally effective at the −10 site in the expression control region; astrong promoter is to be avoided in order to insure complete repressionin the uninduced state. With respect to the −35 region, the sequence ofthe wild-type lac operon, TTTACA, is one base removed from the consensus−35 sequence TTGACA. It is contemplated that a tightly regulable lacoperon-derived expression control region could be constructed using aweaker −35 sequence (i.e., one having less identity with the consensus−35 sequence) and a wild-type −10 sequence (TATAAT), yielding a weakpromoter that needs the assistance of the CAP activator protein.Similarly, it is to be understood that the nucleic acid sequence of theCAP binding region can be altered as long as the CAP protein binds to itwith essentially the same affinity. The spacer region between the end ofthe Shine-Dalgarno sequence and the ATG start of the lacZ codingsequence is typically between about 5 and about 10 nucleotides inlength, preferably about 5 to about 8 nucleotides in length, morepreferably about 7-9 nucleotides in length. The most preferredcomposition and length of the spacer region depends on the compositionand length of Shine-Dalgarno sequence with which it is operably linkedas well as the translation start codon employed (i.e., AUG, GUG, orUUG), and can be determined accordingly by one of skill in the art.Preferably the nucleotide composition of the spacer region is “AT rich”;that is, it contains more A's and T's than it does G's and C's.

In one embodiment of the method of the invention, the expression vectorhas the identifying characteristics of pLAC 11 (ATCC No. 207108) and, ina particularly convenient embodiment, is pLAC 11 (ATCC No. 207108).

As used in the present invention, the term “vector” is to be broadlyinterpreted as including a plasmid, including an episome, a viralvector, a cosmid, or the like. A vector can be circular or linear,single-stranded or double-stranded, and can comprise RNA, DNA, ormodifications and combinations thereof. Selection of a vector or plasmidbackbone depends upon a variety of characteristics desired in theresulting construct, such as selection marker(s), plasmid copy number,and the like. A nucleic acid sequence is “operably linked” to anexpression control sequence in the regulatory region of a vector, suchas a promoter, when the expression control sequence controls orregulates the transcription and/or the translation of that nucleic acidsequence. A nucleic acid that is “operably linked” to an expressioncontrol sequence includes, for example, an appropriate start signal(e.g., ATG) at the beginning of the nucleic acid sequence to beexpressed and a reading frame that permits expression of the nucleicacid sequence under control of the expression control sequence to yieldproduction of the encoded peptide. The regulatory region of theexpression vector optionally includes a termination sequence, such as acodon for which there is no corresponding aminoacetyl-tRNA, thus endingpeptide synthesis. Typically, when the ribosome reaches a terminationsequence or codon during translation of the mRNA, the polypeptide isreleased and the ribosome-mRNA-tRNA complex dissociates.

An expression vector optionally includes one or more selection or markersequences, which typically encode an enzyme capable of inactivating acompound in the growth medium. The inclusion of a marker sequence can,for example, render the host cell resistant to an antibiotic, or it canconfer a compound-specific metabolic advantage on the host cell. Markerssuch as green fluorescent protein also can be used to monitor growth ortoxicity in host cells in which it is expressed. Cells can betransformed with the expression vector using any convenient method knownin the art, including chemical transformation, e.g., whereby cells aremade competent by treatment with reagents such as CaCl₂; electroporationand other electrical techniques; microinjection and the like.

The vector may further include a tightly regulable expression controlsequence operably linked to the nucleic acid sequence encoding thepolypeptide, particularly a stabilized polypeptide, as described herein.In embodiments of the method that use a tightly regulable expressionsystem derived from the lac operon, the host cell is or has beengenetically engineered or otherwise altered to contain a source of Lacrepressor protein in excess of the amount produced in wild-type E. coli.A host cell that contains an excess source of Lac repressor protein isone that expresses an amount of Lac repressor protein sufficient torepress expression of the peptide under repressed conditions, i.e., inthe absence of an inducing agent, such as isopropyl β-D-thiogalactoside(IPTG). Preferably, expression of Lac repressor protein is constitutive.For example, the host cell can be transformed with a second vectorcomprising a gene encoding Lac repressor protein, preferably lacI, morepreferably lacI^(q), to provide an excess source of Lac repressorprotein in trans, i.e., extraneous to the tightly regulable expressionvector. An episome can also serve as a trans source of Lac repressor.Another option for providing a trans source of Lac repressor protein isthe host chromosome itself, which can be genetically engineered toexpress excess Lac repressor protein. Alternatively, a gene encoding Lacrepressor protein can be included on the tightly regulable expressionvector that contains the peptide-encoding oligonucleotide so that Lacrepressor protein is provided in cis. The gene encoding the Lacrepressor protein is preferably under the control of a constitutivepromoter.

The invention is not limited by the type of host cell used forscreening. The host cell can be a prokaryotic or a eukaryotic cell.Preferred mammalian cells include human cells, of any tissue type, andcan include cancer cells or cell lines (e.g., HeLa cells) or otherimmortalized cell lines, hybridomas, pluripotent or omnipotent cellssuch as stem cells or cord blood cells, etc., without limitation.Preferred yeast host cells include Saccharomyces cerevisiae andSchizosaccharomyces pombe. Preferred bacterial host cells include gramnegative bacteria, such as E. coli and various Salmonella spp., and grampositive bacteria, such as bacteria from the genera Staphylococcus,Streptococcus and Enterococcus. Protozoan cells are also suitable hostcells. In clear contrast to conventional recombinant protein expressionsystems, it is preferable that the host cell contains proteases and/orpeptidases, since the selection will, as a result, be advantageouslybiased in favor of peptides that are protease- and peptidase-resistant.More preferably, the host cell has not been modified, genetically orotherwise, to reduce or eliminate the expression of any naturallyexpressed proteases or peptidases. The host cell can be selected with aparticular purpose in mind. For example, if it is desired to obtainpeptide drugs specific to inhibit Staphylococcus, peptides can beadvantageously expressed and screened in Staphylococcus.

There is, accordingly, tremendous potential for the application of thistechnology in the development of new antibacterial peptides useful totreat various pathogenic bacteria. Of particular interest are pathogenicStaphylococci, Streptococci, and Enterococci, which are the primarycauses of nosocomial infections. Many of these strains are becomingincreasingly drug-resistant at an alarming rate. The technology of thepresent invention can be practiced in a pathogenic host cell to isolateinhibitor peptides that specifically target the pathogenic strain ofchoice. Inhibitory peptides identified using pathogenic microbial hostcells in accordance with the invention may have direct therapeuticutility; based on what is known about peptide import, it is very likelythat small peptides are rapidly taken up by Staphylococci, Streptococci,and Enterococci. Once internalized, the inhibitory peptides identifiedaccording to the invention would be expected to inhibit the growth ofthe bacteria in question. It is therefore contemplated that novelinhibitor peptides so identified can be used in medical treatments andtherapies directed against microbial infection. It is furthercontemplated that these novel inhibitor peptides can be used, in turn,to identify additional novel antibacterial peptides using a syntheticapproach. The coding sequence of the inhibitory peptides is determined,and peptides are then chemically synthesized and tested in the host cellfor their inhibitory properties.

Novel inhibitor peptides identified in a pathogenic microbial host cellaccording to the invention can also be used to elucidate potential newdrug targets. The protein target that the inhibitor peptide inactivatedis identified using reverse genetics by isolating mutants that are nolonger inhibited by the peptide. These mutants are then mapped in orderto precisely determine the protein target that is inhibited. Newantibacterial drugs can then be developed using various known or yet tobe discovered pharmaceutical strategies.

Following transformation of the host cell, the transformed host cell isinitially grown under conditions that repress expression of the peptide.Expression of the peptide is then induced. For example, when a lacpromoter/operator system is used for expression, IPTG is added to theculture medium. A determination is subsequently made as to whether thepeptide is inhibitory to host cell growth, wherein inhibition of hostcell growth under induced but not repressed conditions is indicative ofthe expression of a bioactive peptide.

Alternatively, a vector encoding a marker such as green fluorescentprotein (GFP) can be used to monitor toxicity of the random peptides ina host. In general, fluorescence can be monitored in cells that areexpressing both GFP and a randomized peptide and compared with thefluorescence of control cells, i.e., cells expressing only GFP. If therandomized peptide is toxic to the host, fluorescence would not beobserved or would be decreased relative to the control cells.Alternatively, GFP or other markers can be used to monitor the cells forcomplete or partial inhibition of cell division, or for induction ofapoptosis.

For example, to identify a potential anticancer peptide, a cancer cellline such as the HeLa cell line can be used as the host. The cell linecan be transfected with one or more vectors such that the cell lineexpresses both a marker (e.g., GFP) and a peptide from a library ofrandom peptides. It should be noted that the nucleic acid sequencesencoding the marker and the random peptides can be on the same ordifferent vectors. Expression of the random peptides can be controlledby a tightly regulable control sequence, although this need not be thecase. The transfected cells can be seeded into multi-well plates (e.g.,96-well plates) or into multiple flasks, with each well or platereceiving cells collectively expressing a single random peptide. In oneembodiment, the number of cells seeded into each well or flask is chosento ensure that an adequate number of cells expressing a random peptideand a marker is present in each well or flask. This can depend, forexample, on the original transfection efficiency. Under theseconditions, the marker will be observed in all wells or in each flask,unless a peptide that is being expressed is toxic to the cells orotherwise exhibits a desirable bioactivity (e.g., causes a complete orpartial inhibition of cell division, or induces apoptosis). In wells inwhich fluorescence is not observed or the level of fluorescence isdecreased, the random peptides are candidates for anti-cancer peptides.Candidate anti-cancer peptides identified by this method can be furtherscreened to determine if the peptide is selectively toxic or otherwisebioactive in cancer cells. For example, the bioactivity can be comparedbetween malignant and non-malignant cells using a 96-well screeningformat similar to that described above.

In a similar fashion, the method of the invention can be used toidentify peptides that exert an agonist effect on cell division andgrowth. For example, stem cells and cord blood cells, which typically donot proliferate well, can be employed as the host cells. Candidatepeptides can be assayed for a positive effect on cell division andgrowth. Agonistic peptides may be useful in wound healing, organtransplantation and cardiovascular applications.

A plurality of vectors (e.g., a library) that encode a population ofrandomized peptides can be used to identify bioactive peptides (e.g.,antimicrobial or anticancer peptides). A library can include at leasttwo different vectors (e.g., at least five, 50, 500, 5000, 50,000,100,000, 1×10⁶, 5×10⁶ or more vectors), with each of the vectorsencoding different, randomized peptides (e.g., at least five, 50, 500,5000, 50,000, 100,000, 1×10⁶, 5×10⁶ or more different randomizedpeptides). Bioactive peptides can be identified by screening each of therandomized peptides encoded by the different vectors, for a desiredbioactivity (e.g., cell toxicity). Randomized peptides that exhibit thedesired bioactivity can be selected as bioactive peptides.

During development and testing of the intracellular screening method ofthe present invention, it was surprisingly discovered that severalbioactive peptides identified from the randomized peptide library sharedparticular structural features. For example, a disproportionately highnumber of bioactive peptides identified using the intracellularscreening method contained one or more proline residues at or near apeptide terminus. A disproportionate number also contained sequencespredicted, using structure prediction algorithms well-known in the art,to form secondary structures such as a helices or sheets; or ahydrophobic membrane spanning domain. Bioactive fusion proteinscomprising the randomized peptide sequence fused to the Rop protein, dueto a deletion event in the expression vector, were also identified.

Accordingly, randomized peptides used in the screening method of theinvention can be optionally engineered according to the method of theinvention in a biased synthesis to increase their stability by makingone or both of the N-terminal or C-terminal ends more resistant toproteases and peptidases. For example, a vector can include a nucleicacid sequence encoding a stabilized polypeptide, wherein the stabilizedpolypeptide includes a randomized peptide and a stabilizing grouppositioned at the N- and/or C-terminus of the randomized peptide. Theresulting stabilized polypeptide includes the randomized peptide and thestabilizing group coupled to one or both of the randomized peptide'stermini. By “coupled to . . . one or both . . . termini” it is meantthat the randomized peptide is covalently linked, at one or both of itstermini, to the stabilizing group. The nucleic acid sequence thatencodes the randomized peptide in the expression vector or theexpression vector itself is preferably modified such that a firststabilizing group is positioned at the N-terminus of the peptide, and asecond stabilizing group is positioned at the C-terminus of the peptide.

Notably, the bioactive peptides identified according to the method ofthe invention are, by reason of the method itself, stable in theintracellular environment of the host cell. The method of the inventionthus preferably identifies bioactive peptides that are resistant toproteases and peptidases. Resistance to proteases and peptidases can beevaluated by measuring peptide degradation when in contact withappropriate cell extracts (e.g., bacterial, yeast, or human cellextracts), employing methods well-known in the art. A bioactive peptide,without stabilization, can be used as a control. For example,degradation of a stabilized, biotinylated peptide can be assessed byelectrophoresis through an SDS-polyacrylamide gel and Western blottingusing an avidin-horseradish peroxidase conjugate. Alternatively,resistance to proteases and peptidases can be evaluated by measuringpeptide degradation when in contact with purified peptidases and/orproteases (e.g., the Lon and Clp proteases from E. coli). A protease- orpeptidase-resistant peptide exhibits a longer half-life in the presenceof proteases or peptidases compared to a control peptide.

In should be noted that the stabilization of peptides (e.g.,polypeptides containing about 2 to about 50 amino acids) in accordancewith the present invention is an unexpected as peptides, unlikeproteins, are relatively unstable in physiological environments. Forexample, the half-life of most peptides in physiological environments isabout 2 minutes, whereas the half-life of most proteins in physiologicalenvironments is typically well in excess of 2 minutes and is oftenmeasured in hours or days. Proteins possess an inherent stability as aresult of complex intramolecular interactions wherein, due to “proteinfolding” sections of the polypeptide that are distant on the linearchain are close together in space resulting in tertiary and quaternarystructure. Peptides, on the other hand, typically possess, at most, oneor two secondary structural elements (e.g., α-helix, β-sheet or β-turn).Many peptides possess no apparent secondary structural elements at all.

Stabilizing groups are amino acid sequences that can range in size froma single amino acid to a polypeptide (>50 amino acids). Suitablestabilizing groups do not specifically bind to serum proteins (e.g.,albumin) or immunoglobulins, and in many embodiments, are free ofdisulfide bonds. Thus, the stabilizing groups of the present inventiondirectly stabilize the peptides to which they are attached. Stabilizinggroups that do not elicit, or elicit only minimal (i.e., clinicallyacceptable), immune responses in subject mammals are particularlyuseful.

In one embodiment, the stabilizing group is a stable protein, preferablya small stable protein such as thioredoxin, glutathionesulfotransferase, maltose binding protein, glutathione reductase, or afour-helix bundle protein such as Rop protein, as described below,although no specific size limitation on the protein anchor is intended.

Proteins suitable for use as stabilizing groups can be either naturallyoccurring or non-naturally occurring. They can be isolated from anendogenous source, chemically or enzymatically synthesized, or producedusing recombinant DNA technology. Proteins that are particularlywell-suited for use as stabilizing groups are those that are relativelyshort in length and form very stable structures in solution. Proteinshaving molecular weights of less than about 70 kD (e.g., less than about65, 60, 50, 40, 25, or 12 kD) are useful as stabilizing groups. Forexample, human serum albumin has a molecular weight of about 64 kD; E.coli thioredoxin has a molecular weight of about 11.7 kD; E. coliglutathione sulfotransferase has a molecular weight of about 22.9 kD;Rop from the ColE1 replicon has a molecular weight of about 7.2 kD; andmaltose binding protein (without its signal sequence) has a molecularweight of about 40.7 kD. The small size of the Rop protein makes itespecially useful as a stabilizing group, fusion partner, or peptide“anchor”, in that it is less likely than larger proteins to interferewith the accessibility of the linked peptide, thus preserving itsbioactivity. Rop's highly ordered anti-parallel four-helix bundletopology (after dimerization), slow unfolding kinetics (see, e.g., Betzet al, Biochemistry 36, 2450-2458 (1997)), and lack of disulfide bondsalso contribute to its usefulness as a peptide anchor according to theinvention. Other proteins with similar folding kinetics and/orthermodynamic stability (e.g., Rop has a midpoint temperature ofdenaturation, T_(m), of about 71° C., Steif et al., Biochemistry 32,3867-3876 (1993)) are also preferred peptide anchors.

Peptides or proteins having highly stable tertiary structures, such as afour-helix bundle topology as exemplified in Rop, are particularlyuseful. Thus, in another embodiment of the screening method of theinvention, the expression vector encodes a stabilizing group comprisingan α-helical moiety at the N-terminus, C-terminus, or both, of therandomized peptide. The resulting fusion protein is predicted to be morestable than the randomized peptide itself in the host intracellularenvironment. Suitable α-helical moieties can range from a single α-helixto two, three, four, or five α-helix bundles.

Non-limiting examples of single α-helical moieties that can be used tostabilize a bioactive peptide include the following: a 17 amino acidpeptide based on the first α-helix of the α-helix/turn/α-helix peptideof Fezoui et al., Proc. Natl. Acad. Sci. USA 91, 3675-3679 (1994)(Asp-Trp-Leu-Lys-Ala-Arg-Val-Glu-Gln-Glu-Leu-Gln-Ala-Leu-Glu-Ala-Arg,SEQ ID NO:111); an 18 to 36 amino acid peptide containing only glutamicacid, lysine, and glutamine residues, such as (Glu-Lys-Gln)_(y) where yis 6 to 12, although no specific arrangement of the three amino acidswithin the repeating tripeptide is intended; a 20 amino acid peptidecontaining amino acids 14-33 of Neuropeptide Y(Ala-Glu-Asp-Leu-Ala-Arg-Tyr-Tyr-Ser-Ala-Leu-Arg-His-Tyr-Ile-Asn-Leu-Ile-Thr-Arg,SEQ ID NO:112); a 21 amino acid peptide based on amino acids 88-108 ofhuman mannose binding protein(Ala-Ala-Ser-Glu-Arg-Lys-Ala-Leu-Gln-Thr-Glu-Met-Ala-Arg-Ile-Lys-Lys-Ala-Leu-Thr-Ala,SEQ ID NO:113); a 24 amino acid peptide based on amino acids 4-27 ofhelodermin(Ala-Ile-Phe-Thr-Glu-Glu-Tyr-Ser-Lys-Leu-Leu-Ala-Lys-Leu-Ala-Leu-Gln-Lys-Tyr-Leu-Ala-Ser-Ile-Leu,SEQ ID NO:114); and a 34 amino acid peptide based on amino acids 41-74of ribosomal protein L9(Pro-Ala-Asn-Leu-Lys-Ala-Leu-Glu-Ala-Gln-Lys-Gln-Lys-Glu-Gln-Arg-Gln-Ala-Ala-Glu-Glu-Leu-Ala-Asn-Ala-Lys-Lys-Leu-Lys-Glu-Gln-Leu-Glu-Lys,SEQ ID NO: 115).

Non-limiting examples of two-helix bundles include two α-helicesconnected by a turn region (see, for example, the 38 amino acidα-helix/turn/α-helix peptide of Fezoui et al. (1994), supra); a 42 aminoacid peptide based on amino acids 512-553 of the adhesion modulationdomain (AMD) of α-catenin; a 64 amino acid peptide based on residues411-475 of α-catenin; and a 78 amino acid a peptide based on residues24-102 of seryl-tRNA synthetase from E. coli.

Non-limiting examples of three-helix bundles include a peptide based onresidues 410-504 of α-catenin, a (Gly-Pro-Pro-)₁₀ or (-Pro-Pro-Gly)₁₀peptide, and an (Ala-Pro-Pro-)₁₀ or (-Pro-Pro-Ala)₁₀ peptide.

Two-helix bundles may dimerize and form a four-helix bundle. Asindicated above, Rop, which is 63 amino acids in size, forms a two-helixbundle that automatically dimerizes to become a four-helix bundle. Otheruseful four-helix bundles include the 35 amino acid and 51 amino acidfour-helix bundle peptides of Betz et al. (1997) supra, and the 125amino acid AMD of the α-catenin protein (residues 509-633 of theα-catenin protein).

Where a small stable protein or an α-helical moiety, such as afour-helix bundle protein, is fused to the N-terminus, the randomizedpeptide can optionally be further stabilized by, for example, covalentlylinking one or more prolines, with or without a following undefinedamino acids (e.g., -Pro, -Pro-Pro, -Pro-Xaa_(n), -Pro-Pro-Xaa_(n), etc.)at the C-terminus of the peptide sequence, where n is 1 or 2; likewise,when the α-helical moiety is fused to the C-terminus, the randomizedpeptide can be further stabilized by, for example, covalently linkingone or more prolines, with or without a preceding undefined amino acid(e.g., Pro-, Pro-Pro-, Xaa_(n)-Pro-, Xaa_(n)-Pro-Pro-, etc.) at theN-terminus of the peptide sequence, where n is 1 or 2, as discussed inmore detail below.

In another embodiment of the screening method of the invention, thestabilizing group can constitute one or more proline (Pro) residues.Preferably, a proline dipeptide (Pro-Pro) is used as a stabilizinggroup, although additional prolines may be included. The encodedproline(s) are typically naturally occurring amino acids. However, ifand to the extent a proline derivative, for example a hydroxyproline ora methyl- or ethyl-proline derivative, can be encoded or otherwiseincorporated into the peptide, those proline derivatives are also usefulas stabilizing groups.

At the N-terminus of the peptide, the stabilizing group also can be anoligopeptide having the sequence Xaa_(n)-Pro_(m)-, wherein Xaa is anyamino acid (e.g., Ala), m is greater than 0, and n is 1 or 2. Forexample, m can be about 1 to about 5 (e.g., m can be 2 or 3). Anoligopeptide having the sequence Xaa_(n)-Pro_(m)-, wherein m=2, isparticularly useful. Likewise, at the C-terminus of the peptide, thestabilizing group can be an oligopeptide having the sequence-Pro_(m)-Xaa_(n), wherein Xaa is any amino acid (e.g., Ala), m isgreater than 0, and n is 1 or 2. For example, m can be about 1 to about5 (e.g., m can be 2 or 3). An oligopeptide having the sequencePro_(m)-Xaa_(n), wherein m=2, is particularly useful.

In one embodiment of the screening method of the invention, the nucleicacid sequence that encodes the randomized peptide in the expressionvector is modified to encode both a first stabilizing group linked tothe N-terminus of the peptide, the first stabilizing group beingselected from the group consisting of small stable protein, Pro-,Pro-Pro-, Xaa_(n)-Pro-, and Xaa_(n)-Pro-Pro-, and a second stabilizinggroup linked to the C-terminus of the peptide, the second stabilizinggroup being selected from the group consisting of a small stableprotein, -Pro, -Pro-Pro, Pro-Xaa_(n) and Pro-Pro-Xaa_(n). The resultingpeptide has enhanced stability in the intracellular environment relativeto a peptide lacking the terminal stabilizing groups.

In yet another embodiment of the screening method of the invention, theputative bioactive peptide is stabilized by engineering into the peptidea stabilizing motif such as an α-helix motif or an opposite chargeending motif. Chemical synthesis of an oligonucleotide according to thescheme [(CAG)A(TCAG)] yields an oligonucleotide encoding a peptideconsisting of a random mixture of the hydrophilic amino acids H is, Gln,Asn, Lys, Asp, and Glu (see Table 14). Except for aspartate, these aminoacids are most often associated with α-helical secondary structuralmotifs; the resulting oligonucleotides are thus biased in favor ofoligonucleotides that encode peptides that are likely to form α-helicesin solution.

Alternatively, the putative bioactive peptide is stabilized by flankinga randomized region with a region of uniform charge (e.g., positivecharge) on one end and a region of opposite charge (e.g., negative) onthe other end, to form an opposite charge-ending motif. To this end, thenucleic acid sequence that encodes the randomized peptide in theexpression vector or the expression vector itself is preferably modifiedto encode a plurality of sequential uniformly charged amino acids at theN-terminus of the peptide, and a plurality of sequential oppositelycharged amino acids at the C-terminus of the peptide. The positivecharges are supplied by a plurality of positively charged amino acidsconsisting of lysine, histidine, arginine or a combination thereof; andthe negative charges are supplied by a plurality of negatively chargedamino acids consisting of aspartate, glutamate or a combination thereof.It is expected that such a peptide will be stabilized by the ionicinteraction of the two oppositely charged ends. Preferably, the putativebioactive peptide contains at least three charged amino acids at eachend. More preferably, it contains at least four charged amino acids ateach end. In a particularly preferred embodiment, the larger acidicamino acid glutamate is paired with the smaller basic amino acid lysine,and the smaller acidic amino acid aspartate is paired with the largerbasic amino acid arginine.

The present invention further provides a bioactive peptide containingone or more structural features or motifs selected to enhance thestability of the bioactive peptide in an intracellular environment. Forexample, a bioactive peptide of the invention can include anystabilizing group as described above in connection with the screeningmethod of the invention. Thus, stabilized bioactive peptides identifiedusing the screening method of the invention are included in theinvention. Likewise, both known bioactive peptides and bioactivepeptides subsequently discovered, when linked to one or more stabilizinggroups as described herein, are also within the scope of the presentinvention.

Accordingly, the invention provides a bioactive peptide having astabilizing group at its N-terminus, its C-terminus, or at both termini.

The bioactive peptide of the invention includes a bioactive peptide thathas been detectably labeled, derivatized, or modified in any mannerdesired prior to use, provided it contains one or more terminalstabilizing groups as provided herein. For example, a non-stabilizingmoiety (e.g., a label) can be attached to either terminus of thebioactive peptide, which terminus may or may not also include astabilizing group.

The stabilized bioactive peptide of the invention can be synthesizedenzymatically, chemically, or produced by recombinant geneticengineering, without limitation, as described in more detail below. Inany synthetic peptide having a stabilizing group that includes one ormore prolines according to the present invention, the proline ispreferably a naturally occurring amino acid; alternatively, however, itcan be a synthetic derivative of proline, for example a hydroxyprolineor a methyl- or ethyl-proline derivative. Accordingly, where theabbreviation “Pro” is used herein in connection with a stabilizing groupthat is part of a synthetic peptide, it is meant to include prolinederivatives in addition to a naturally occurring proline.

In a bioactive peptide stabilized at only one terminus (i.e., at eitherthe N- or the C-terminus), the stabilizing group is preferably anα-helical moiety (e.g., four-helix bundle protein such as Rop protein),or one or more proline residues, with or without an undefined amino acid(Xaa). The resulting polypeptide consists essentially of a bioactivepeptide and the stabilizing group coupled to one terminus of thebioactive peptide.

A peptide stabilized at both termini can include a first stabilizinggroup attached to the N-terminus, and a second stabilizing groupattached to the C-terminus, where the first and second stabilizinggroups are as defined previously in connection with the method foridentifying bioactive peptides. The stabilizing group is covalentlyattached to the peptide (e.g., via a peptide bond).

In one embodiment of the bioactive peptide of the invention, the firststabilizing group is Xaa_(n)-Pro_(m)-, with or without a precedingundefined amino acid (e.g., Pro-, Pro-Pro-, Xaa_(n)-Pro-,Xaa_(n)-Pro-Pro-, etc.), and the second stabilizing group is-Pro_(m)-Xaa_(n), with or without a following undefined amino acids(e.g., -Pro, -Pro-Pro, -Pro-Xaa_(n), -Pro-Pro-Xaa_(n), etc.). In anotherembodiment, the first (N-terminal) stabilizing group is a small stableprotein or an α-helical moiety (e.g., a four-helix bundle protein suchas Rop protein); and the second (C-terminal) stabilizing group is-Pro_(m)-Xaa_(n) or one or more proline residues (e.g., -Pro-Pro). Inyet another embodiment, the second (C-terminal) stabilizing group is asmall stable protein or an α-helical moiety (e.g., a four-helix bundleprotein such as Rop protein) and the first (N-terminal) stabilizinggroup is Xaa_(n)-Pro_(m)- or one or more proline residues.

The invention further provides a peptide stabilized by flanking theamino acid sequence of a bioactive peptide with an opposite chargeending motif, as described herein. Preferably, the resulting stabilizedpeptide retains at least a portion of the biological activity of thebioactive protein. The stabilized peptide includes a peptide that hasbeen detectably labeled, derivatized, or modified in any manner desiredprior to use.

It should be understood that any bioactive peptide, without limitation,can be stabilized according to the invention by attaching a stabilizinggroup to either or both of the N- and C-termini. Included in the presentinvention are various antimicrobial peptides, inhibitory peptides,therapeutic peptide drugs, and the like. Non-limiting examples includeadrenocorticotropic hormone, bactericidal/permeability-increasingprotein (BPI), brain natriuretic peptide, cercropin, endothelin,pentagastrin, scorpion peptides, teriparatide acetate, and all of thepeptides listed in Tables 1 and 2, that have been modified at one orboth peptide termini to include a stabilizing group as discussed above.Particularly useful bioactive peptides include insulin, glucagon,calcitonin, somatostatin, gonadotrophin, and secretin.

The invention is exemplified by peptides such asPro-Pro-Asp-Arg-Val-Tyr-Ile-His-Pro-Phe-His-Ile-Pro-Pro (SEQ ID NO:3)andGlu-Asp-Glu-Asp-Asp-Arg-Val-Tyr-Ile-His-Pro-Phe-His-Ile-Arg-Lys-Arg-Lys(SEQ ID NO:4), wherein the middle nine amino acids(Asp-Arg-Val-Tyr-Ile-His-Pro-Phe-His-Ile-; SEQ ID NO: 5) constitute thesequence of angiotensin. In embodiments in which the bioactive peptideis a known peptide (e.g., angiotensin), the stabilizing group or groupsthat are coupled to one or both of the bioactive peptide's termini arenot naturally associated with the peptide. In other words, thestabilizing groups that are coupled to the bioactive peptide areheterologous to the bioactive peptide.

In embodiments in which a first stabilizing group is coupled to theN-terminus of a bioactive peptide and a second stabilizing group iscoupled to the C-terminus of the bioactive peptide, the first and secondstabilizing groups can be the same or different. In some embodimentswhere the first and second stabilizing groups are different, they can besaid to be “heterologous” to each other, i.e., the stabilizing groupshave different amino acid sequences and (1) are from different proteins,or (2) are from the same protein, but the stabilizing groups are notcontiguous with each other in a naturally occurring protein, or (3) areproduced synthetically and one or both of the stabilizing groups do notcorrespond to a naturally occurring sequence. For example, a bioactivepeptide can be coupled to Rop at the N-terminus and coupled to atwo-helix bundle from the α-catenin AMD protein on the C-terminus.

Where the first and second stabilizing groups are from the same protein,it is not necessary that these flanking groups interact with each otherso as to confine or constrain the N-terminus and C-terminus of theflanked peptide in close proximity to one another. For example, Lavallieand others (Bio/Technology 11:187-193 (1993); McCoy et al., U.S. Pat.Nos. 5,270,181; 5,292,646; 5,646,016 and 6,143,524) describe internalpeptide fusions at the active site loop of thioredoxin, citing theadvantages of strong secondary structure in that region, the absence oftertiary structure, and constraint of the peptide at both ends. In theinternal thioredoxin fusion, the inserted peptide is bound at each endby cysteine residues, which may form a disulfide linkage and furtherlimit the conformational freedom of the inserted peptide.

However, the present work suggests that neither steric constraint of thepeptide ends nor any of the other unique characteristics of thioredoxinpolypeptides are necessary. Proteins other than thioredoxin, such asRop, can be effectively used as first and/or second stabilizing groupsfor bioactive peptides. Neither the first nor the second stabilizinggroup needs to possess the capacity to participate in the formation ofan intramolecular disulfide bond. That is, the stabilizing group doesnot need to contain a cysteine or, if it does, the remainder of thepolypeptide need not contain a cysteine. The stabilizing groups can beselected such that disulfide formation between the first and secondstabilizing groups, if it occurs at all, does not bring the N-terminusand C-terminus of the bioactive peptide into close proximity. Forexample, flanking stabilizing groups can be selected such that they donot contain, within about three residues of the ends that are connectedto the ends of the bioactive peptide, cysteine residues that interactwith each other to form an intramolecular disulfide bond.

Moreover, in embodiments in which the first and second stabilizinggroups are from the same polypeptide, the stabilizing groups need not,although they may, interact to from a naturally occurring secondary ortertiary structure. Naturally occurring secondary structures include,for example, α-helices, β-sheets, β-turns and the like that are presentin, for example, the native solution or crystal structure of the proteinas determined by X-ray crystallography or nuclear magnetic resonancespectroscopy. Naturally occurring tertiary structures result from the“folding” of sections the polypeptide that may be distant on the linearchain such that they are close together in space. Tertiary structureincludes the three-dimensional relationships between and among thesecondary structures and unstructured portions of the molecule.

Modification of a bioactive peptide to yield a stabilized bioactivepeptide according to the invention can be achieved by standardtechniques well-known in the arts of genetics and peptide synthesis. Forexample, where the peptide is synthesized de novo, as in solid statepeptide synthesis, one or more prolines or other stabilizing groups canbe added at the beginning and the end of the peptide chain during thesynthetic reaction. In recombinant synthesis, for example as describedin Example III herein, one or more codons encoding proline, or codonsencoding α-helical moieties, for example, can be inserted into thepeptide coding sequence at the beginning and/or the end of the sequence,as desired. Preferably, codons encoding N-terminal prolines are insertedafter (i.e., 3′ to) the initiation site ATG (which encodes methionine).Analogous techniques are used to synthesize bioactive peptides having anopposite charge ending motif. When a known bioactive peptide is modifiedto yield a stabilized bioactive peptide according to the invention, theunmodified peptide can conveniently be used as a control in a protease-or peptidase-resistance assay as described hereinabove to confirm, ifdesired, that the modified peptide exhibits increased stability.

A stabilized bioactive peptide according to the invention can include apeptide whose bioactivity is evident from or identified in a “phagedisplay” experiment. In “phage display” peptides are displayed on thesurface of phage and assayed for bioactivity. Displayed peptides aretethered at the one terminus, typically the C-terminus, to thebacteriophage surface. Their other terminus, typically the N-terminus,is free (i.e., non-fused). Structurally, the polypeptide produced in aphage display system is typically a fusion polypeptide that contains apeptide of interest at the N-terminus, followed by a phage protein atthe C-terminus. In some phage display polypeptides, however, the orderis reversed and the phage protein is at the N-terminus of thepolypeptide and the peptide of interest is at the C-terminus. The phageprotein is selected such that the polypeptide is displayed on thesurface of the bacteriophage. Examples include bacteriophage proteinspIII and pVIII.

Phage display can be used to screen peptide libraries and identify novelbioactive peptides. Bioactive peptides that are active when displayedtypically continue to exhibit bioactivity when fabricated synthetically(i.e., without fusion to the phage protein), but they frequently exhibitinstability in vivo. This may be due to the fact that the C-terminus isno longer protected or tethered. Hence, the present invention includes amethod for stabilizing a bioactive peptide by linking a stabilizinggroup to the N-terminus or C-terminus of a peptide, when the bioactivepeptide has been identified using phage display.

Additionally or alternatively, the genetic constructs used to producethe fusion protein within the bacteriophage can be engineered encode astabilizing group at the terminus of encoded fusion polypeptide thatwould otherwise have been free, such that the fusion polypeptidedisplayed on the surface of the bacteriophage contains a peptide ofinterest flanked by a stabilizing group at the terminus and abacteriophage protein at the other terminus.

The invention thus includes methods for phage display of stabilizedbioactive proteins, methods for stabilizing bioactive peptidesidentified using phage display, and bioactive peptides thus identifiedand/or stabilized.

The present invention also provides a cleavable polypeptide comprising astabilized, bioactive peptide either immediately preceded by (i.e.,adjacent to the N-terminus of the bioactive peptide) a cleavage site, orimmediately followed by (i.e., adjacent to the C-terminus of thebioactive peptide) a cleavage site. Thus, a bioactive peptide ascontemplated by the invention can be part of a cleavable polypeptide.The cleavable polypeptide is cleavable, either chemically, as withcyanogen bromide, or enzymatically, to yield the bioactive peptide. Theresulting bioactive peptide either includes a first stabilizing grouppositioned at its N-terminus and/or a second stabilizing grouppositioned at its C-terminus, both as described hereinabove. Thecleavage site immediately precedes the N-terminal stabilizing group orimmediately follows the C-terminal stabilizing group. In the case of abioactive peptide stabilized with an opposite charge ending motif, thecleavage site immediately precedes the first charged region orimmediately follows the second charged region. The cleavage site makesit possible to administer a bioactive peptide in a form that could allowintracellular targeting and/or activation.

Alternatively, a bioactive peptide of the invention can be fused to anoncleavable N-terminal or C-terminal targeting sequence wherein thetargeting sequence allows targeted delivery of the bioactive peptide,e.g., intracellular targeting or tissue-specific targeting of thebioactive peptide. In one embodiment of this aspect of the invention, astabilizing group (e.g., one or more proline residues) is positioned atthe free (i.e., non-fused) terminus of the bioactive peptide asdescribed hereinabove in connection with the screening method foridentifying bioactive peptides. The targeting sequence attached to theother peptide terminus can, but need not, contain a small stable proteinsuch as Rop or one or more proline residues, as long as the targetingfunction of the targeting sequence is preserved. In another embodimentof this aspect of the invention, the bioactive peptide is stabilizedwith a charge ending motif as described hereinabove, wherein one chargedregion is coupled to the free terminus of the bioactive peptide, and theother charged region is disposed between the targeting sequence and theactive sequence of the bioactive peptide.

The invention further includes a method for using an antimicrobialpeptide that includes covalently linking a stabilizing group, asdescribed above, to the N-terminus, the C-terminus, or to both termini,to yield a stabilized antimicrobial peptide, then contacting a microbewith the stabilized antimicrobial peptide. Alternatively, the stabilizedantimicrobial peptide used in this aspect of the invention is made bycovalently linking oppositely charged regions, as described above, toeach end of the antimicrobial peptide to form an opposite charge endingmotif. An antimicrobial peptide is to be broadly understood as includingany bioactive peptide that adversely affects a microbe such as abacterium, virus, protozoan, or the like, as described in more detailabove. An example of an antimicrobial peptide is an inhibitory peptidethat inhibits the growth of a microbe. When the antimicrobial peptide iscovalently linked to a stabilizing group at only one peptide terminus,any of the stabilizing groups described hereinabove can be utilized.When the antimicrobial peptide is covalently linked to a stabilizinggroup at both peptide termini, the method includes covalently linking afirst stabilizing group to the N terminus of the antimicrobial peptideand a second stabilizing group to the C terminus of the antimicrobialpeptide, where the first and second stabilizing groups are as definedpreviously in connection with the method for identifying bioactivepeptides. In a preferred embodiment of the method for using anantimicrobial peptide, one or more prolines, more preferably a Pro-Prodipeptide, is attached to at least one, preferably both, termini of theantimicrobial peptide. Alternatively, or in addition, anXaa_(n)-Pro_(m)-sequence, as described above, can be attached to theN-terminus of a microbial peptide, and/or a -Pro_(m)-Xaa_(n) sequencecan be attached to the C-terminus, to yield a stabilized antimicrobialpeptide.

The antimicrobial peptide thus modified in accordance with the inventionhas enhanced stability in the intracellular environment relative to anunmodified antimicrobial peptide. As noted earlier, the unmodifiedpeptide can conveniently be used as a control in a protease- orpeptidase-resistance assay as described hereinabove to confirm, ifdesired, that the modified peptide exhibits increased stability.Further, the antimicrobial activity of the antimicrobial peptide ispreferably preserved or enhanced in the modified antimicrobial peptide;modifications that reduce or eliminate the antimicrobial activity of theantimicrobial peptide are easily detected and are to be avoided.

The invention further provides a method for inhibiting the growth of amicrobe comprising contacting the microbe with a stabilized inhibitorypeptide. As described above, the stabilized inhibitory peptide can havea stabilizing group attached at its N-terminus, C-terminus, or bothtermini.

Also included in the present invention is a method for treating apatient having a condition treatable with a peptide drug, comprisingadministering to the patient a peptide drug that has been stabilized asdescribed herein. Peptide drugs for use in therapeutic treatments arewell known (see, e.g., Table 1). However, they are often easily degradedin biological systems, which affects their efficacy. In one embodimentof the present method, the patient is treated with a stabilized drugcomprising the peptide drug of choice and a stabilizing group linked toeither the N-terminus, the C-terminus of, or to both termini of thepeptide drug. In another embodiment of the present method, the patientis treated with a stabilized drug comprising the peptide drug of choicethat has been stabilized by attachment of oppositely charged regions toboth termini of the peptide drug. Because the peptide drug is therebystabilized against proteolytic degradation, greater amounts of the drugshould reach the intended target in the patient.

In embodiments of the method involving administration of a peptide drugthat is covalently linked to a stabilizing group at only one peptideterminus, the stabilizing group is preferably an α-helical moiety, suchas a four-helix bundle protein (e.g., Rop), provided that attachment ofthe α-helical moiety to the peptide terminus preserves a sufficientamount of efficacy for the drug. It is to be nonetheless understood thatthe group or groups used to stabilize the peptide drug are as definedhereinabove, without limitation. In embodiments involving administrationof a peptide drug covalently linked to a stabilizing group at bothpeptide termini, the peptide drug includes a first stabilizing grouplinked to the N-terminus of the peptide drug and a second stabilizinggroup linked to the C-terminus of the peptide drug. Thus, in anotherpreferred embodiment of the treatment method of the invention, thestabilized peptide drug includes one or more prolines, more preferably aproline-proline dipeptide, attached to one or both termini of thepeptide drug. For example, the peptide drug can be stabilized bycovalent attachment of a Rop protein at one terminus, and by covalentattachment of a proline or proline dipeptide at the other terminus; inanother preferred embodiment, the peptide drug can be stabilized byproline dipeptides at each of the N-terminus and C terminus.Alternatively, or in addition, the stabilized peptide drug used in thetreatment method can include an Xaa_(n)-Pro_(m)-sequence at theN-terminus of the peptide drug, and/or a -Pro_(m)-Xaa_(n) sequence atthe C-terminus. Optionally, prior to administering the stabilizedpeptide drug, the treatment method can include covalently linking astabilizing group to one or both termini of the peptide drug to yieldthe stabilized peptide drug.

If desired, the unmodified peptide drug can conveniently be used as acontrol in a protease- or peptidase-resistance assay as describedhereinabove to confirm that the stabilized peptide drug exhibitsincreased stability. Further, the therapeutic efficacy of the peptidedrug is preferably preserved or enhanced in the stabilized peptide drug;modifications that reduce or eliminate the therapeutic efficacy of thepeptide drug are easily detected and are to be avoided.

The present invention further includes a fusion protein comprising afour-helix bundle protein, preferably Rop protein, and a polypeptide.Preferably the polypeptide is bioactive; more preferably it is abioactive peptide. The fusion protein of the invention can be used inany convenient expression vector known in the art for expression oroverexpression of a peptide or protein of interest. Optionally, acleavage site is present between the four-helix bundle protein and thepolypeptide to allow cleavage, isolation and purification of thepolypeptide. In one embodiment of the fusion protein, the four-helixbundle protein is covalently linked at its C-terminus to the N-terminusof the polypeptide; in an alternative embodiment, the four-helix bundleprotein is covalently linked at its N-terminus to the C-terminus of thepolypeptide. Fusion proteins of the invention, and expression vectorscomprising nucleic acid sequences encoding fusion proteins wherein thenucleic acid sequences are operably linked to a regulatory controlelement such as a promoter, are useful for producing or overproducingany peptide or protein of interest.

EXAMPLES

The present invention is illustrated by the following examples. It is tobe understood that the particular examples, materials, amounts, andprocedures are to be interpreted broadly in accordance with the scopeand spirit of the invention as set forth herein.

Example I Construction and Characterization of a Highly RegulableExpression Vector, pLAC11, and its Multipurpose Derivatives, pLAC22 andpLAC33

A number of different expression vectors have been developed over theyears to facilitate the production of proteins in E. coli and relatedbacteria. Most of the routinely employed expression vectors rely on laccontrol in order to overproduce a gene of choice. While these vectorsallow for overexpression of the gene product of interest, they are leakydue to changes that have been introduced into the lac control region andgene expression can never be shut off under repressed conditions, asdescribed in more detail below. Numerous researchers have noticed thisproblem with the more popular expression vectors pKK223-3 (G. Posfai etal. Gene. 50: 63-67 (1986); N. Scrutton et al., Biochem J. 245: 875-880(1987)), pKK233-2 (P. Beremand et al., Arch Biochem Biophys. 256: 90-100(1987); K. Ooki et al., Biochemie. 76: 398-403 (1994)), pTrc99A (S.Ghosh, Protein Expr. Purif. 10: 100-106 (1997); J. Ranie et al., Mol.Biochem. Parasitol. 61: 159-169 (1993)), as well as the pET series (M.Eren et al., J. Biol. Chem. 264: 14874-14879 (1989); G. Godson, Gene100: 59-64 (1991)).

The expression vector described in this example, pLAC11, was designed tobe more regulable and thus more tightly repressible when grown underrepressed conditions. This allows better regulation of cloned genes inorder to conduct physiological experiments. pLAC11 can be used toconduct physiologically relevant studies in which the cloned gene isexpressed at levels equal to that obtainable from the chromosomal copyof the gene in question. The expression vectors described here weredesigned utilizing the wild-type lac promoter/operator in order toaccomplish this purpose and include all of the lac control region,without modification, that is contained between the start of the O3auxiliary operator through the end of the O1 operator. As with all lacbased vectors, the pLAC11 expression vector described herein can beturned on or off by the presence or absence of the gratuitous inducerIPTG. In experiments in which a bacterial cell contained both a nullallele in the chromosome and a second copy of the wild-type allele onpLAC11 cells grown under repressed conditions exhibited the nullphenotype while cells grown under induced conditions exhibited thewild-type phenotype. Thus the pLAC11 vector truly allows for the gene ofinterest to be grown under either completely repressed or fully inducedconditions. Two multipurpose derivatives of pLAC11, pLAC22 and pLAC33were also constructed to fulfill different experimental needs.

The vectors pLAC11, pLAC22 and pLAC33 were deposited with the AmericanType Culture Collection (ATCC), 10801 University Blvd., Manassas, Va.,20110-2209, USA, on Feb. 16, 1999, and assigned ATCC deposit numbersATCC 207108, ATCC 207110 and ATCC 207109, respectively. It isnonetheless to be understood that the written description herein isconsidered sufficient to enable one skilled in the art to fully practicethe present invention. Moreover, the deposited embodiment is intended asa single illustration of one aspect of the invention and is not to beconstrued as limiting the scope of the claims in any way.

Materials and Methods

Media. Minimal M9 media (6 g disodium phosphate, 3 g potassiumphosphate, 1 g ammonium chloride, 0.5 g sodium chloride, distilled waterto 1 L; autoclave; add 1 mL m magnesium sulfate (1M) and 0.1 mL calciumchloride (1M); a sugar added to a final concentration of 0.2%; vitaminsand amino acids as required for non-prototrophic strains) and rich LBmedia (10 g tryptone, 5 g yeast extract, 10 g sodium chloride, distilledwater to 1 L; autoclave) were prepared as described by Miller (J.Miller, “Experiments in molecular genetics” Cold Spring HarborLaboratory, Cold Spring Harbor, N.Y. (1972). The antibiotics ampicillin,kanamycin, streptomycin, and tetracycline (Sigma Chemical Company, St.Louis, Mo.) were used in rich media at a final concentration of 100, 40,200, and 20 ug/ml, respectively. When used in minimal media,tetracycline was added at a final concentration of 10 îg/ml.5-bromo-4-chloro-3-indoyl β-D-galactopyranoside (Xgal) was added tomedia at a final concentration of 40 îg/ml unless otherwise noted. IPTGwas added to media at a final concentration of 1 mM.

Chemicals and Reagents. When amplified DNA was used to construct theplasmids that were generated in this study, the PCR reaction was carriedout using native Pfu polymerase from Stratagene (Cat. No. 600135). Xgaland IPTG were purchased from Diagnostic Chemicals Limited.

Bacterial Strains and Plasmids. Bacterial strains and plasmids arelisted in Table 4. To construct ALS225, ALS224 was mated with ALS216 andstreptomycin resistant, blue recombinants were selected on a Rich LBplat that contained streptomycin, Xgal, and IPTG. To construct ALS226,ALS224 was mated with ALS217 and streptomycin resistant, kanomycinresistant recombinants were selected on a Rich LB plate that containedstreptomycin and kanamycin. To construct ALSS15, ALSS14 was mated withALS216 and streptomycin resistant, blue recombinants were selected on aRich LB plate that contained streptomycin, Xgal, and IPTG. To constructALS527, ALS524 was mated with ALS224 and streptomycin resistant,tetracycline resistant recombinants were selected on a Rich LB platethat contained streptomycin and tetracycline. To construct ALS535,ALS533 was mated with ALS498 and tetracycline resistant recombinantswere selected on a Minimal M9 Glucose plate that contained tetracycline,leucine and thiamine (B₁) (Sigma Chemical Company). To construct ALS533,a P1 lysate prepared from E. coli strain K5076 (H. Miller et al., Cell20: 711-719 (1980)) was used to transduce ALS224 and tetracyclineresistant transductants were selected.

TABLE 4 Bacterial strains and plasmids used in Example I E. coli StrainsLaboratory Original Name Name Genotype Source ALS216 SE9100 araD139Δ(lac)U169 thi E. Altman et al., J. Biol. flbB5301 deoC7 ptsF25 Chem.265: 18148-18153 rpsE/F′ lacl^(q) ^(l) Z⁺ Y⁺ A⁺ (1990) ALS217 SE9100.1araD139 Δ(lac)U169 thi S. Emr (Univ. of California, flbB5301 deoC7ptsF25 San Diego) rpsE/F′ lacl^(q) ^(l) Z::Tn5 Y+ A+ ALS221 BL21(DE3)ompT hsdS(b) (R-M-) gal F. Studier et al., J. Mol. Biol. dcm 189:113-130 (1986) ALS224 MC1061 araD139 Δ(araABOIC- M. Casadaban et al., J.Mol. leu) 7679 Δ(lac)X74 galU Biol. 138: 179-207 (1980) galK rpsL hsr−hsm+ ALS225 MC1061/F′ lacl^(q) ^(l) Z⁺ Y⁺ This example A⁺ ALS226MC1061/F′ lacl^(q) ^(l) Z::Tn5 This example Y⁺ A⁺ ALS269 CSH27 F-trpA33thi J. Miller, “Experiments in molecular genetics” Cold SpringLaboratory, Cold Spring Harbor, NY (1972) ALS413 MG1655 E. coliwild-type F-λ- M. Guyer et al., Cold Spring Harbor Symp. Quant. Biol.45: 135-140 (1980) ALS498 JM101 supE thi Δ(lac-proAB)/ C. Yanisch-Perronet al., F′ traD36proA⁺B⁺ lacl^(q) Gene. 33: 103-119 (1985) Δ(lacZ) M15ALS514 NM554 MC1061 recA13 E. Raleigh et al., Nucl. Acids Res. 16:1563-1575 (1988) ALS515 MC1061 recA13/F′ This example lacl^(q) ^(l) Z⁺Y⁺ A⁺ ALS524 XL1-Blue recAI endAI gyrA96 thi-I Stratagene (Cat. No.200268) hsdRI7 supE44 relAI lac/F′ proAB lacl^(q) Δ(lacZ) M15 Tn10ALS527 MC1061/F′ proAB lacl^(q) This example Δ(lacZ) M15 Tn10 ALS533MC1061 proAB::Tn10 This example ALS535 MC1061 proAB::Tn10/ This exampleF′ lacl^(q) Δ(lacZ) M15 proA+B+ ALS598 CAG18615 zjb-3179::Tn10dKan M.Singer et al., Microbiol. lambda-rph-1 Rev. 53: 1-24 (1989) PlasmidsPlasmid Name Relevant Characteristics Source pBH20 wild-type lacpromoter/ K. Itakura et al., Science. 198: 1056-1063 operator, Amp^(R),Tet^(R), colE1 (1977) replicon pBR322 Amp^(R), Tet^(R), colE1 repliconF. Bolivar et al., Gene. 2: 95-113 (1977) pET-21(+) T7 promoter/lacoperator, lacIq, Novagen (Cat. No. 69770-1) Amp^(R), colE1 repliconpGE226 wild-type recA gene, Amp^(R) J. Weisemann, et al., J. Bacteriol.163: 748-755 (1985) pKK223-3 tac promoter/operator, Amp^(R), J. Brosiuset al., Proc. Natl. Acad. Sci. colE1 replicon USA 81: 6929-6933 (1984)pKK223-2 trc promoter/operator, Amp^(R), E. Amann et al., Gene. 40:183-190 colE1 replicon (1985) pLysE T7 lysozyme, Cam^(R), P15A F.Studier, J. Mol. Biol. 219: 37-44 replicon (1991) pLysS T7 lysozyme,Cam^(R), P15A F. Studier, J. Mol. Biol. 219: 37-44 replicon (1991)pMS421 wild-type lac promoter/ D. Graña et al., Genetics. 120: 319-327operator, lacIq, Strep^(R), Spec^(R), (1988) SC101 replicon pTer7wild-type lacZ coding region, R. Young (Texas A&M University) Amp^(R)pTrc99A trc promoter/operator, lacIq, E. Amann et al., Gene. 69: 301-315Amp^(R), colE1 replicon (1988) pUC8 lac promoter/operator, Amp^(R), J.Vieira et al., Gene. 19: 259-268 colE1 replicon (1982) pXE60 wild-typeTOL pWWO xylE J. Westpheling (Univ. of Georgia) gene, Amp^(R)

Construction of the pLAC11, pLAC22, and pLAC33 expression vectors. Toconstruct pLAC11, primers #1 and #2 (see Table 5) were used topolymerase chain reaction (PCR) amplify a 952 base pair (bp fragmentfrom the plasmid pBH20 which contains the wild-type lac operon. Primer#2 introduced two different base pair mutations into the seven basespacer region between the Shine Dalgarno site and the ATG start site ofthe lacZ which converted it from AACAGCT to AAGATCT thus placing a BglII site in between the Shine Dalgarno and the start codon of the lacZgene. The resulting fragment was gel isolated, digested with Pst I andEcoR I, and then ligated into the 3614 bp fragment from the plasmidpBR322ΔAvaI which had been digested with the same two restrictionenzymes. To construct pBR322ΔAva1, pBr₃₂₂ was digested with Ava1, filledin using Klenow, and then religated. To construct pLAC22, a 1291 bp NcoI. EcoR I fragment was gel isolated from pLAC21 and ligated to a 4361 bpNco I. EcoR I fragment which was gel isolated from pBR322/NcoI. Toconstruct pLAC21, primers #2 and #3 (see Table 5) were used to PCRamplify a 1310 bp fragment from the plasmid pMS421 which contains thewild-type lac operon as well as the lacI^(q) repressor. The resultingfragment was gel isolated, digested with EcoR I, and then ligated intopBR322 which had also been digested with EcoR I. To construct pBR322/NcoI, primers #4 and #5 (see Table 5) were used to PCR amplify a 788 bpfragment from the plasmid pBR322. The resulting fragment was gelisolated, digested with Pst I and EcoR I, and then ligated into the 3606bp fragment from the plasmid pBR322 which had been digested with thesame two restriction enzymes. The pBR322Mco I vector also contains addedKpn 1 and Sma I sites in addition to the new Nco I site. To constructpLAC33, a 2778 bp fragment was gel isolated from pLAC12 which had beendigested with BsaB I and Bsa I and ligated to a 960 bp fragment frompUC8 which had been digested with Afl III, filled in with Klenow, andthen digested with Bsa I. To construct pLAC12, a 1310 bp Pst I, BamH Ifragment was gel isolated from pLAC11 and ligated to a 3232 bp Pst I,BamH I fragment which was gel isolated from pBR322.

TABLE 5 Primers employed to PCT amplify DNA fragments that were used inthe construction of the various plasmids described in Example I

In Table 5 the regions of the primers that are homologous to the DNAtarget template are indicated with a dotted underline, while therelevant restriction sites are indicated with a solid underline. Allprimers are listed in the 5′→3′ orientation.

Compilation of the DNA sequences for the pLAC11, pLAC22, and pLAC33expression vectors. All of the DNA that is contained in the pLAC11,pLAC22, and, pLAC33 vectors has been sequenced.

The sequence for the pLAC11 vector, which is 4547 bp, can be compiled asfollows: bp 1-15 are AGATCTTATGAATTC (SEQ ID NO: 20) from primer #2(Table 5); bp 16-1434 are bp 4-1422 from pBR322 (GenBank Accession #JO1749); bp 1435-1442 are TCGGTCGG, caused by filling in the Ava I site inpBR322AAvaI; bp 1443-4375 is bp 1427-4359 from pBR322 (GenBank Accession#JO1749); and bp 4376-4547 are bp 1106-1277 from the wild-type E. colilac operon (GenBank Accession #J01636).

The sequence for the pLAC22 vector which is 5652 bp can be compiled asfollows: bp 1-15 are AGATCTTATGAATTC(SEQ ID NO: 21) from primer #2(Table 5); bp 16-4370 are bp 4-4358 from pBR322 (GenBank Accession#J01749); bp 4371-4376 is CCATGG which is the Nco I site from pBR322/NcoI; and bp 4377-5652 are bp 2-1277 from the wild-type E. coli lac operon(GenBank Accession #J01636), except that bp #4391 of the pLAC22 sequenceor bp#16 from the wild-type E. coli lac operon sequence has been changedfrom a ″C″ to a ″T″ to reflect the presence of the lacI^(q) mutation (J.Brosius et al., Proc. Natl. Acad. Sci. USA. 81: 6929-6933 (1984)).

The sequence for the pLAC33 vector which is 3742 bp can be compiled asfollows: bp 1-15 is AGATCTTATGAATTC (SEQ. ID NO: 22) from primer #2(Table 5); bp 16-1684 are bp 4-1672 from pEk322 (GenBank Accession #501749); bp 1685-2638 are bp 786-1739 from pUC8 (GenBank Accession#L09132); bp 2639-3570 are bp 3428-4359 from pBR322 (GenBank Accession#30 J01749); and bp 3571-3742 are bp 1106-1277 from the wild-type E.coli lac operon (GenBank Accession #J01636). In the maps for thesevectors, the ori is identified as per Balbás (P. Balbás et al., Gene.50: 3-40 (1986)), while the lacPO is indicated starting with the O3auxiliary operatic and ending with the O1 operator as per Müller-Hill(B. Müller-Hill, The lac Operon: A Short History of a Genetic Paradigm.Walter de Gruyter, Berlin, Germany (1996)).

Construction of the pLAC11-, pLAC22-, pLAC33-, pKK223-3-, pKK233-2-,pTrc99A-, and pET-21(+)-lacZ constructs. To construct pLAC11-lacZ,pLAC22-lacZ, and pLAC33-lacZ, primers #6 and #7 (see Table 5) were usedto PCR amplify a 3115 bp fragment from the plasmid pTer7 which containsthe wild-type lacZ gene. The resulting fragment was gel isolated,digested with Bgl II and Hind III, and then ligated into the pLAC11,pLAC22 or pLAC33 vectors that had been digested with the same tworestriction enzymes. To construct pKK223-3-lacZ and pKK233-2-lacZ,primers #8 and #9 (see Table 5) were used to PCR amplify a 3137 bpfragment from the plasmid pTer7. The resulting fragment was gelisolated, digested with Pst I and Hind III, and then ligated into thepKK223-3 or pKK233-2 vectors which had been digested with the same tworestriction enzymes. To construct pTrc99A-lacZ and pET-21 (+)-lacZ,primers #9 and #10 (see Table 5) were used to PCR amplify a 3137 bpfragment from the plasmid pTer7. The resulting fragment was gelisolated, digested with BamH I and Hind III, and then ligated into thepTrc99A or pET-21 (+) vectors which had been digested with the same tworestriction enzymes.

Construction of the pLAC11-recA and xylE constructs. To constructpLAC11-recA, primers #11 and #12 (see Table 5) were used to PCR amplifya 1085 bp fragment from the plasmid pGE226 which contains the wild-typerecA gene. The resulting fragment was gel isolated, digested with Bgl IIand Hind III, and then ligated into the pLAC11 vector which had beendigested with the same two restriction enzymes. To constructpLAC11-xylE, primers #13 and #14 (see Table 5) were used to PCR amplifya 979 bp fragment from the plasmid pXE60 which contains the wild-typePseudomonas putida xylE gene isolated from the TOL pWW0 plasmid. Theresulting fragment was gel isolated, digested with Bgl II and EcoR I,and then ligated into the pLAC11 vector which had been digested with thesame two restriction enzymes.

Assays. β-galactosidase assays were performed as described by Miller (J.Miller. “Experiments in molecular genetics,” Cold Spring HarborLaboratory, Cold Spring Harbor, N.Y. (1972)), while catechol2,3-dioxygenase (catO₂ase) assays were performed as described byZukowski, et. al. (M. Zukowski et al., Proc. Natl. Acad. Sci. U.S.A. 80:1101-1105 (1983)).

Results

Construction and features of pLAC11, pLAC22, and pLAC33. Plasmid mapsthat indicate the unique restriction sites, drug resistances, origin ofreplication, and other relevant regions that are contained in pLAC11,pLAC22, and pLAC33 are shown in FIGS. 2, 3 and 4, respectively. pLAC11was designed to be the most tightly regulable of these vectors. Itutilizes the ColE1 origin of replication from pBR322 and Lac1 repressoris provided in trans from either an episome or another compatibleplasmid. pLAC22 is very similar to pLAC11, however, it also containslacI^(q), thus a source of LacI does not have to be provided in trans.pLAC33 is a derivative of pLAC11 which utilizes the mutated ColE1 originof replication from pUC8 (S. Lin-Chao et al., Mol. Micro. 6: 3385-3393(1992)) and thus pLAC33's copy number is significantly higher thanpLAC11 and is comparable to that of other pUC vectors. Because thecloning regions of these three vectors are identical, cloned genes canbe trivially shuffled between and among them depending on the expressiondemands of the experiment in question.

To clone into pLAC 11, pLAC22, or pLAC33, PCR amplification is performedwith primers that are designed to introduce unique restriction sitesjust upstream and downstream of the gene of interest. Usually a Bgl IIsite is introduced immediately in front of the ATG start codon and anEcoR I site is introduced immediately following the stop codon. Anadditional 6 bases is added to both ends of the oligonucleotide in orderto ensure that complete digestion of the amplified PCR product willoccur. After amplification the double-stranded (ds) DNA is restrictedwith Bgl II and EcoR I, and cloned into the vector which has also beenrestricted with the same two enzymes. If the gene of interest contains aBlgII site, then BamH I or Bcl I can be used instead since they generateoverhangs which are compatible with Bgl II. If the gene of interestcontains an EcoR I site, then a site downstream of EcoR I in the vector(such as Hind III) can be substituted.

Comparison of pLAC11, pLAC22, and pLAC33, to other expression vectors.In order to demonstrate how regulable the pLAC11, pLAC22, and pLAC33expression vectors were, the wild-type lacZ gene was cloned into pLAC11,pLAC22, pLAC33, pKK223-3, pKK233-2, pTrc99A, and pET-21(+). Constructswhich required an extraneous source of Lac1 for their repression weretransformed into ALS225, while constructs which contained a source ofLac1 on the vector were transformed into ALS224. pET-21 (+) constructswere transformed into BL21 because they require T7 RNA polymerase fortheir expression. Four clones were chosen for each of these sevenconstructs and β-galactosidase assays were performed under repressed andinduced conditions. Rich Amp overnights were diluted 1 to 200 in eitherRich Amp Glucose or Rich Amp IPTG media and grown until they reachedmid-log(OD₅₅₀=0.5). In the case of PET-21(+) the pLysE and pLysSplasmids, which make T7 lysozyme and thus lower the amount of availableT7 polymerase, were also transformed into each of the constructs. Table6 shows the results of these studies and also lists the induction ratiothat was determined for each of the expression vectors. As the dataclearly indicate, pLAC 11 is the most regulable of these expressionvectors and its induction ratio is close to that which can be achievedwith the wild-type lac operon. The vector which yielded the lowest levelof expression under repressed conditions was pLAC 11, while the vectorwhich yielded the highest level of expression under induced conditionswas pLAC33.

TABLE 6 β-galactosidase levels obtained in different expression vectorsgrown under either repressed or induced conditions # of Miller UnitsObserved Repressed Induced Fold Vector Source Conditions ConditionsInduction pLAC11 F′ 19 11209 590X pLAC22 Plasmid 152 13315  88X pLAC33F′ 322 23443  73X pKK223-3 F′ 92 11037 120X pKK233-2 F′ 85 10371 122XpTrc99A Plasmid 261 21381  82X pET-21(+) Plasmid 2929 16803  6XpET-21(+)/pLysE Plasmid 4085 19558  5X pET-21(+)/pLysS Plasmid 159820268  13XThe average values obtained for the four clones that were tested fromeach vector are listed in the table. Standard deviation is not shown butwas less than 5% in each case. Induction ratios are expressed as theratio of enzymatic activity observed at fully induced conditions versusfully repressed conditions. The plasmid pLysE yielded unexpectedresults; it was expected to cause lower amounts of lacZ to be expressedfrom pET-21(+) under repressed conditions and, instead, higher amountswere observed. As a result, both pLysE and pLysS were restriction mappedto make sure that they were correct.Demonstrating that pLAC11 constructs can be tightly regulated. pLAC11was designed to provide researchers with an expression vector that couldbe utilized to conduct physiological experiments in which a cloned geneis studied under completely repressed conditions where it is off orpartially induced conditions where it is expressed at physiologicallyrelevant levels. FIG. 5 demonstrates how a pLAC11-lacZ construct can beutilized to mimic chromosomally expressed lacZ that occurs under variousphysiological conditions by varying the amount of IPTG inducer that isadded. ALS226 cells containing pLAC11-lacZ were grown to mid-log in richmedia that contained varying amounts of IPTG and then β-galactosidaseactivity was assayed. Also indicated in the graph are the averageβ-galactosidase activities obtained for strains with a singlechromosomal copy of the wild-type lacZ gene that were grown underdifferent conditions.

To demonstrate just how regulable pLAC11 is, the recA gene was clonedinto the pLAC11 vector and transformed into cells which contained a nullrecA allele in the chromosome. As the results in Table 7 clearly show,recombination cannot occur in a host strain which contains anonfunctional RecA protein and thus P1 lysates which provide a Tn10dKantransposon cannot be used to transduce the strain to Kan^(R) at a highfrequency. recA⁻ cells which also contain the pLAC11-recA construct canbe transduced to Kan^(R) at a high frequency when grown under inducedconditions but cannot be transduced to Kan^(R) when grown underrepressed conditions.

TABLE 7 The recombination (−) phenotype of a recA null mutant strain canbe preserved with a pLAC11-recA (wild-type) construct under repressedconditions Repressed Conditions Induced Conditions Number of Kan^(R)Number of Kan^(R) Strain transductants transductants ALS225 (recA⁺)178,000 182,000 ALS514 (recA⁻) 5 4 ALS515 (recA⁻ 4 174,000 pLAC11-recA)

The data presented in Table 7 are the number of Kan^(R) transductantsthat were obtained from the different MC1061 derivative strains whenthey were transduced with a P1 lysate prepared from strain ALS598 whichharbored a Tn10dKan transposon insertion. Overnights were prepared fromeach of these strains using either rich medium to which glucose wasadded at a final concentration of 0.2% (repressed conditions) or richmedium to which IPTG was added at a final concentration of 1 mM (inducedconditions). The overnights were then diluted 1 to 10 into the samemedium which contained CaCl₂ added to a final concentration of 10 mM andaerated for two hours to make them competent for transduction with P1phage. Cells were then spectrophotometrically normalized and 0.1 mlaliquots of cells at an OD₅₅₀ of 5 were transduced with 0.1 ml ofconcentrated P1 lysate as well as 0.1 ml of P1 lysates that had beendiluted to 10⁻¹, 10⁻², or 10⁻³. 0.2 ml of 0.1 M Sodium Citrate was addedto the cell/phage mixtures and 0.2 ml of the final mixtures were platedonto Rich Kanamycin plates and incubated overnight at 37° C. The totalnumber of Kan^(R) colonies were then counted. ALS225 recA⁺ data pointswere taken from the transductions which used the 10⁻³ diluted phage,while ALS514 recA⁻ data points were taken from the transductions whichused the concentrated phage. The data points for ALS515 recA⁻pCyt-3-recA grown under repressed conditions were taken from thetransductions which used the concentrated phage, while the data pointsfor ALS515 recA⁻ pCyt-3-recA grown under induced conditions were takenfrom the transductions which used the 10⁻³ diluted phage.

Testing various sources of Lac for trans repression of pLAC11. BecausepLAC11 was designed to be used with an extraneous source of LacIrepressor, different episomal or plasmid sources of LacI which areroutinely employed by researchers were tested. Since one of the LacIsources also contained the lacZ gene, a reporter construct other thanpLAC11-lacZ was required and thus a pLAC11-xylE construct wasengineered. Table 8 shows the results of this study.

All of the LacI sources that were tested proved to be adequate torepress expression from pLAC11, however, some were better than others.The basal level of expression that was observed with F's which providedlacI^(q1) or with the plasmid pMS421 which provided lacI^(q) atapproximately six copies per cell was lower than the basal level ofexpression that was observed with F's which provided lacI^(q) all threetimes that the assay was run. Unfortunately, however, the xylE genecould not be induced as high when lacI^(q1) on a F′ or lacI^(q) on aplasmid was used as the source of Lac repressor.

TABLE 8 Catechol 2,3-dioxygenase levels obtained for a pLAC11-xylEconstruct when Lac repressor is provided by various sources Catechol2,3-dioxygenase activity in milliunits/mg Repressed Induced StrainSource of LacI Conditions Conditions ALS224 None 32.7 432.8 ALS535F′lacIq Δ(lacZ)M15 .3 204.4 proA+B+ Tn10 ALS527 F′lacIq Δ(lacZ)M15 .3243.3 proA+B+ ALS227 pMS421 lacI^(q) .2 90.9 ALS225 F′lacIq^(l) Z⁺ Y⁺ A⁺.2 107.4 ALS226 F′lacIq^(l) Z::Tn5 Y⁺ .2 85.1 A⁺The wild-type xylE gene was cloned into the pLAC11 vector and theresulting pLAC11-xylE construct was then transformed into each of theMC1061 derivative strains listed in the table. Rich overnights werediluted 1 to 200 in either Rich Glucose or Rich IPTG media and grownuntil they reached mid-log(OD₅₅₀=0.5). Cell extracts were then preparedand catechol 2,3-dioxygenase assays were performed as described byZukowski, et. al. (Proc. Natl. Acad. Sci. U.S.A. 80:1101-1105 (1983)).The average values obtained in three different experiments are listed inthe table. Standard deviation is not shown but was less 15 than 10% ineach case.Discussion

Most of the routinely employed expression vectors rely on lac control inorder to overproduce a gene of choice. The lac promoter/operatorfunctions as it does due to the interplay of three main components.First, the wild-type lac-10 region (TATGTT) is very weak. c-AMPactivated CAP protein is able to bind to the CAP site just upstream ofthe −35 region which stimulates binding of RNA polymerase to the weak−10 site. Repression of the lac promoter is observed when glucose is themain carbon source because very little c-AMP is present which results inlow amounts of available c-AMP activated CAP protein. When poor carbonsources such as lactose or glycerol are used, c-AMP levels rise andlarge amounts of c-AMP activated CAP protein become available. Thusinduction of the lac promoter can occur. Second, Lac repressor binds tothe lac operator. Lac repressor can be overcome by allolactose which isa natural byproduct of lactose utilization in the cell, or by thegratuitous inducer, IPTG. Third, the lac operator can form stable loopstructures which prevents the initiation of transcription due to theinteraction of the Lac repressor with the lac operator (O1) and one oftwo auxiliary operators, O2 which is located downstream in the codingregion of the lacZ gene, or O3 which is located just upstream of the CAPbinding site.

While binding of Lac repressor to the lac operator is the major effectorof lac regulation, the other two components are not dispensable.However, most of the routinely used lac regulable vectors either containmutations or deletions which alter the affect of the other twocomponents. The pKK223-3 (J. Brosius et al., Proc. Natl. Acad. Sci. USA.81:6929-6933 (1984)), pKK233-2 (E. Amann et al., Gene. 40:183-190(1985)), pTrc99A (E. Amann et al., Gene. 69:301-315 (1988)), and pETfamily of vectors (F. Studier, Method Enzymol. 185:60-89 (1990)) containonly the lac operator (O1) and lack both the CAP binding site as well asthe O3 auxiliary operator. pKK223-3, pKK233-2, and pTrc99 use a trp-lachybrid promoter that contains the trp-35 region and the lacUV5-10 regionwhich contains a strong TATAAT site instead of the weak TATGTT site. ThepET family of vectors use the strong T7 promoter. Given thisinformation, perhaps it is not so surprising researchers have found itis not possible to tightly shut off genes that are cloned into thesevectors.

The purpose of the studies described in Example I was to design a vectorwhich would allow researchers to better regulate their cloned genes inorder to conduct physiological experiments. The expression vectorsdescribed herein were designed utilizing the wild-type lacpromoter/operator in order to accomplish this purpose and include all ofthe lac control region, without modification, that is contained betweenthe start of the O3 auxiliary operator through the end of the O1operator. As with all lac based vectors, the pLAC11, pLAC22, and pLAC33expression vectors can be turned on or off by the presence or absence ofthe gratuitous inducer IPTG.

Because the new vector, pLAC11, relies on the wild-type lac controlregion from the auxiliary lac O3 operator through the lac O1 operator,it can be more tightly regulated than the other available expressionvectors. In direct comparison studies with pKK-223-3, pKK233-2, pTrc99A,and pET-21(+), the lowest level of expression under repressed conditionswas achievable with the pLAC11 expression vector. Under fully inducedconditions, pLAC11 expressed lacZ protein that was comparable to thelevels achievable with the other expression vectors. Induction ratios of1000× have been observed with the wild-type lac operon. Of all theexpression vectors that were tested, only pLAC11 yielded inductionratios which were comparable to what has been observed with thewild-type lac operon. It should be noted that the regulation achievableby pLAC11 may be even better than the data in Table 6 indicates. BecauselacZ was used in this test, the auxiliary lac O₂ operator which residesin the coding region of the lacZ gene was provided to the pKK223-3,pKK233-2, pTrc99A, and pET-21(+) vectors which do not normally containeither the O2 or O3 auxiliary operators. Thus the repressed states thatwere observed in the study in Table 6 are probably lower than one wouldnormally observe with the pKK223-3, pKK233-2, pTrc99A, and pET-21(+)vectors.

To meet the expression needs required under different experimentalcircumstances, two additional expression vectors which are derivativesof pLAC11 were designed. pLAC22 provides lacIq on the vector and thusunlike pLAC11 does not require an extraneous source of LacI for itsrepression. pLAC33 contains the mutated ColE1 replicon from pUC8 andthus allows proteins to be expressed at much higher levels due to theincrease in the copy number of the vector. Of all the expressions thatwere evaluated in direct comparison studies, the highest level ofprotein expression under fully induced conditions was achieved using thepLAC33 vector. Because the cloning regions are identical in pLAC11,pLAC22, and pLAC33, genes that are cloned into one of these vectors canbe trivially subcloned into either of the other two vectors depending onexperimental circumstances. For physiological studies, pLAC11 is thebest suited of the three vectors. If, however, the bacterial strain ofchoice can not be modified to introduce elevated levels of Lac repressorprotein which can be achieved by F's or compatible plasmids that providelacI^(q) or lacI^(q1), the pLAC22 vector can be utilized. If maximaloverexpression of a gene product is the goal, then the pLAC33 vector canbe utilized.

Numerous experiments call for expression of a cloned gene product atphysiological levels; i.e., at expression levels that are equivalent tothe expression levels observed for the chromosomal copy of the gene.While this is not easily achievable with any of the commonly utilizedexpression vectors, these kinds of experiments can be done with thepLAC11 expression vector. By varying the IPTG concentrations, expressionfrom the pLAC11 vector can be adjusted to match the expression levelsthat occur under different physiological conditions for the chromosomalcopy of the gene. In fact, strains which contain both a chromosomal nullmutation of the gene in question and a pLAC11 construct of the genepreserve the physiological phenotype of the null mutation underrepressed conditions.

Because the use of Lac repressor is an essential component of anyexpression vector that utilizes the lac operon for its regulation, theability of different source of LacI to repress the pLAC11 vector wasalso investigated. Researchers have historically utilized eitherlacI^(q) constructs which make 10 fold more Lac repressor than wild-typelacI or lacI^(q1) constructs which make 100 fold more Lac repressor thanwild-type lacI (B. Müller-Hill, Prog. Biophys. Mol. Biol. 30:227-252(1975)). The greatest level of repression of pLAC11 constructs could beachieved using F's which provided approximately one copy of thelacI^(q1) gene or a multicopy compatible plasmid which providedapproximately six copies of the lacI^(q) gene. However, the inductionthat was achievable using these lacI sources was significantly lowerthan what could be achieved when F's which provided approximately onecopy of the lacI^(q1) gene were used to repress the pLAC11 construct.Thus if physiological studies are the goal of an investigation, then F'swhich provide approximately one copy of the lacI^(q1) gene or amulticopy compatible plasmid which provides approximately six copies ofthe lacI^(q) gene can be used to regulate the pLAC11 vector. However, ifmaximal expression is desired, then F's which provide approximately onecopy of the lacI^(q) gene can be utilized. Alternatively, if a bacterialstrain can tolerate prolonged overexpression of an expressed gene andoverexpression of a gene product is the desired goal, then maximalexpression under induced conditions is obtained when a bacteria strainlacks any source of Lac repressor.

Example II An In Vivo Approach for Generating Novel Bioactive Peptidesthat Inhibit the Growth of E. coli

A randomized oligonucleotide library containing sequences capable ofencoding peptides containing up to 20 amino acids was cloned into pLAC11(Example I) which allowed the peptides to either be tightly turned offor overproduced in the cytoplasm of E. coli. The randomized library wasprepared using a [NNN] codon design instead of either the [NN(G,T)] or[NN(G,C)] codon design used by most fusion-phage technology researchers.[NN(G,T)] or [NN(G,C)] codons have been widely used instead of [NNN]codons to eliminate two out of the three stop codons, thus increasingthe amount of full-length peptides that can be synthesized without astop codon (J. Scott et al., Science 249:386-390 (1990); J. Delvin etal., Science 249:404-406 (1990); S. Cwirla et al., Proc. Nat'l. Acad.Sci. U.S.A. 87:6378-6382 (1990)). However, the [NN(G,T)] and [NN(G,C)]oligonucleotide codon schemes eliminate half of the otherwise availablecodons and, as a direct result, biases the distribution of amino acidsthat are generated. Moreover, the [NN(G,T)] and [NN(G,C)] codon schemesdrastically affect the preferential codon usage of highly expressedgenes and removes a number of the codons which are utilized by theabundant tRNAs that are present in E. coli (H. Grosjean et al., Gene.18: 199-209 (1982); T. Ikemura, J. Mol. Biol. 151:389-409 (1981)).

Of the 20,000 peptides screened in this Example, 21 inhibitors of cellgrowth were found which could prevent the growth of E. coli on minimalmedia. The top twenty inhibitor peptides were evaluated for strength ofinhibition, and the putative amino acid sequences of the top 10“anchorless” inhibitor peptides were examined for commonly sharedfeatures or motifs.

Materials and Methods

Media. Rich LB and minimal M9 media used in this study was prepared asin Example I. Ampicillin was used in rich media at a final concentrationof 100 îg/ml and in minimal media at a final concentration of 50 îg/ml.IPTG was added to media at a final concentration of 1 mM.

Chemicals and Reagents. Extension reactions were carried out usingKlenow from New England Biolabs while ligation reactions were performedusing T4 DNA Ligase from Life Sciences. IPTG was obtained fromDiagnostic Chemicals Limited.

Bacterial Strains and Plasmids. ALS225, which isMC1061/F′lacI^(q1)Z+Y+A+ (see Example I), was the E. coli bacterialstrain used in this Example. The genotype for MC1061 isaraD139Δ(araABOIC-leu)7679Δ(lac)X74 galU galK rpsL hsr− hsml+ (M.Casadaban et al., J. Mol. Biol. 138: 179-207 (1980)). pLAC11, a highlyregulable expression vector, is described in Example I.

Generation of the Randomized Peptide Library. The 93 baseoligonucleotide 5′TAC TAT AGA TCT ATG (NNN)₂₀ TAA TAA GAA TTC TCG ACA 3′(SEQ ID NO:23), where N denotes an equimolar mixture of the nucleotidesA, C, G, or T, was synthesized with the trityl group and subsequentlypurified with an OPC cartridge using standard procedures. Thecomplementary strand of the 93 base oligonucleotide was generated by anextension/fill-in reaction with Klenow using an equimolar amount of the18 base oligonucleotide primer 5′ TGT CGA GAA TTC TTA TTA 3′ (SEQ IDNO:24). After extension, the resulting ds-DNA was purified using aPromega DNA clean-up kit and restricted with EcoR 1 and Bgl II (Promega,Madison, Wis.). The digested DNA was again purified using a Promega DNAclean-up kit and ligated to pLAC11 vector which had been digested withthe same two restriction enzymes. The resulting library was transformedinto electrocompetent ALS225 E. coli cells under repressed conditions(LB, ampicillin, plus glucose added to 0.2%).

Screening of Transformants to Identify Inhibitor Clones. Transformantswere screened to identify any that could not grow on minimal media whenthe peptides were overproduced. Using this scheme, any transformantbacterial colony that overproduces a peptide that inhibits theproduction or function of a protein necessary for growth of thattransformant on minimal media will be identified. Screening on minimalmedia, which imposes more stringent growth demands on the cell, willfacilitate the isolation of potential inhibitors from the library. It iswell known that growth in minimal media puts more demands on a bacterialcell than growth in rich media as evidenced by the drastically reducedgrowth rate; thus a peptide that adversely affects cell growth is morelikely to be detected on minimal media. Screening was carried out usinga grid-patching technique. Fifty clones at a time were patched onto botha rich repressing plate (LB Amp glucose) and a minimal inducing plate(M9 glycerol Amp IPTG) using an ordered grid. Patches that do not groware sought because presumably these represent bacteria that are beinginhibited by the expressed bioactive peptide. To verify that all of theinhibitors were legitimate, plasmid DNA was made from each inhibitoryclone (QIA Prep Spin Miniprep kit; Qiagen Cat. No. 27104) andtransformed into a fresh background (ALS225 cells), then checked toconfirm that they were still inhibitory on plates and that theirinhibition was dependent on the presence of the inducer, IPTG.

Growth Rate Analysis in Liquid Media. Inhibition strength of thepeptides was assessed by subjecting the inhibitory clones to a growthrate analysis in liquid media. To determine the growth rate inhibition,starting cultures of both the peptides to be tested and a control strainwhich contains pLAC11 were diluted from a saturated overnight culture toan initial OD₅₅₀ of ˜0.01. All cultures were then induced with 1 mM IPTGand OD₅₅₀ readings were taken until the control culture reached an OD₅₅₀of ˜0.5. The hypothetical data in Table 9 shows that when the controlstrain reaches an OD₅₅₀ of about 0.64 (at about 15 hours), a strainwhich contains a peptide that inhibits the growth rate at 50% will onlyhave reached an OD₅₅₀ of only about 0.08. Thus, the growth of a 50%inhibited culture at 15 hours (i.e., the OD₅₅₀ at 15 hours, which isproportional to the number of cells in a given volume of culture) isonly about 12.5% (that is, 0.08/0.64×100) of that of a control strainafter the same amount of time, and the inhibitor peptide would thus haveeffectively inhibited the growth of the culture (as measured by theOD₅₅₀ at the endpoint) by 87.5% (=100%−12.5%).

TABLE 9 Hypothetical data from a peptide that inhibits growth rate at30%, 50% and 70% OD550 readings on a culture which contains a 0D550readings on a peptide that inhibits Time in control culture which thegrowth rate at . . . Hours contains pLAC11 25% 50% 75% 0 .010 .010 .010.010 2.5 .020 .017 .015 .012 5 .040 .028 .020 .014 7.5 .080 .047 .030.017 10 .160 .079 .040 .020 12.5 .320 .133 .060 .024 15 .640 .226 .080.028

An example is shown in FIG. 6, wherein ALS225 cells containing thepLAC11 vector (control), and either the one day inhibitor pPep1 or thetwo day inhibitor pPep12 (see below), were grown in minimal M9 glycerolmedia with IPTG added to 1 mM. OD₅₅₀ readings were then taken hourlyuntil the cultures had passed log phase. Growth rates were determined bymeasuring the spectrophotometric change in OD₅₅₀ per unit time withinthe log phase of growth. The inhibition of the growth rate was thencalculated for the inhibitors using pLAC11 as a control.

Sequencing the Coding Regions of the Inhibitor Peptide Clones. Theforward primer 5′ TCA TTA ATG CAG CTG GCA CG 3′ (SEQ ID NO:25) and thereverse primer 5′ TTC ATA CAC GGT GCC TGA CT 3′ (SEQ ID NO:26) were usedto sequence both strands of the top ten “anchorless” inhibitor peptideclones identified by the grid-patching technique.

If an error-free consensus sequence could not be deduced from these twosequencing runs, both strands of the inhibitor peptide clones inquestion were resequenced using the forward primer 5′ TAG CTC ACT CATTAG GCA CC 3′ (SEQ ID NO:27) and the reverse primer 5′ GAT GAC GAT GAGCGC ATT GT 3′ (SEQ ID NO:28). The second set of primers were designed toanneal downstream of the first set of primers in the pLAC11 vector.

Generating Antisense Derivatives of the Top Five “Anchorless” InhibitorClones.

Oligonucleotides were synthesized which duplicated the DNA insertcontained between the Bgl II and EcoR I restriction sites for the topfive “anchorless” inhibitor peptides as shown in Table 12 with one majornucleotide change. The “T” of the ATG start codon was changed to a “C”which resulted in an ACG which can not be used as a start codon. Theoligonucleotides were extended using the same 18 base oligonucleotideprimer that was used to build the original peptide library. Theresulting ds-DNA was then restricted, and cloned into pLAC11 exactly asdescribed in the preceding section “Generating the randomized peptidelibrary.” The antisense oligonucleotides that were used are as follows:

pPep1(antisense): (SEQ ID NO: 29)5′TAC TAT AGA TCT ACG GTG ACT GAA TTT TGT GGC TTGTTG GAC CAA CTG CCT TAG TAA TAG TGG AAG GCT GAAATT AAT AAG AAT TCT CGA CA 3′; pPepS(antisense): (SEQ ID NO: 30)5′TAC TAT AGA TCT ACG TGG CGG GAC TCA TGG ATT AAGGGT AGG GAC GTG GGG TTT ATG GGT TAA AAT AGT TTGATA ATA AGA ATT CTC GAC A 3′ pPep12(antisense): (SEQ ID NO: 31)5′TAC TAT AGA TCT ACG AAC GGC CGA ACC AAA CGA ATCCGG GAC CCA CCA GCC GCC TAA ACA GCT ACC AGC TGTGGT AAT AAG AAT TCT CGA CA 3′ pPep13(antisense): (SEQ ID NO: 32)5′TAC TAT AGA TCT ACG GAC CGT GAA GTG ATG TGT GCGGCA AAA CAG GAA TGG AAG GAA CGA ACG CCA TAG GCCGCG TAA TAA GAA TTC TCG ACA 3′ pPep19(antisense): (SEQ ID NO: 33)5′TAC TAT AGA TCT ACG AGG GGC GCC AAC TAA GGG GGGGGG AAG GTA TTT GTC CCG TGC ATA ATC TCG GGT GTTGTC TAA TAA GAA TTC TCG ACA 3′Results

Identifying and Characterizing Inhibitor Peptides from the Library.Approximately 20,000 potential candidates were screened as describedhereinabove, and 21 IPTG-dependent growth inhibitors were isolated. Allthe inhibitors so identified were able to prevent the growth of the E.coli bacteria at 24 hours, and three of the 21 inhibitors were able toprevent the growth of the E. coli bacteria at 48 hours, using the gridpatching technique. These three inhibitors were classified as “two day”inhibitors; the other 18 were classified as “one-day” inhibitors.

Results from the growth rate analysis for candidate peptide inhibitorsare shown in Table 10. The % inhibition of the growth rate wascalculated by comparing the growth rates of cells that contained inducedpeptides with the growth rate of cells that contained the induced pLAC11vector. Averaged values of three independent determinations are shown.

TABLE 10 Ability of the Inhibitor Peptides to Inhibit Cell Growth %Inhibitor Type Inhibition pLAC11 — 0 (control) pPep1 1 Day 25 pPep2 1Day 23 pPep3 2 Day 80 pPep4 1 Day 21 pPep5 1 Day 24 pPep6 1 Day 27 pPep71 Day 26 pPep8 1 Day 29 pPep9 1 Day 22 pPep10 1 Day 24 pPep11 1 Day 22pPep12 2 Day 82 pPep13 1 Day 28 pPep14 2 Day 71 pPep15 1 Day 23 pPep16 1Day 24 pPep17 1 Day 28 pPep18 1 Day 24 pPep19 1 Day 29 pPep20 1 Day 19pPep21 1 Day 23

Of the 21 peptides that were tested, the one-day inhibitor peptidesinhibited the bacterial growth rate at a level of approximately 25%,while the two-day inhibitor peptides inhibited the bacterial growth rateat levels greater than 75%. As can be seen from the hypothetical data inTable 9, a one-day inhibitor which inhibited the growth rate at 25%would have only reached an OD₅₅₀ of 0.226 when the control strainreached an OD₅₅₀ of 0.64. At that point in time, the growth of theculture that is inhibited by a one-day inhibitor (as measured by theend-point OD₅₅₀) only be only 35.3% of that of a control strain at thatpoint; thus the inhibitor peptide would have effectively inhibited thegrowth of the culture by 64.7%. A two-day inhibitor which inhibited thegrowth rate at 75% would have only reached an OD₅₅₀ of 0.028 when thecontrol strain reached an OD₅₅₀ of 0.64. Thus the growth of the culturethat is being inhibited by a two-day inhibitor will only be 4.4% of thatof the control strain at this point, and the inhibitor peptide wouldhave effectively inhibited the growth of the culture by 95.6%. Thesecalculations are consistent with the observation that two-day inhibitorsprevent the growth of bacteria on plates for a full 48 hours while theone-day inhibitors only prevent the growth of bacteria on plates for 24hours.

All 21 candidates were examined using restriction analysis to determinewhether they contained 66 bp inserts as expected. While most of themdid, the two-day inhibitors pPep3 and pPep14 were found to contain ahuge deletion. Sequence analysis of these clones revealed that thedeletion had caused the carboxy-terminal end of the inhibitor peptidesto become fused to the amino-terminal end of the short 63 amino acid Ropprotein. The rop gene, which is part of the ColE1 replicon, is locateddownstream from where the oligonucleotide library is inserted into thepLAC11 vector.

Sequence Analysis of the Top 10 “Anchorless” Inhibitor Peptides. The DNAfragments comprising the sequences encoding the top 10 “anchorless”inhibitor peptides (i.e., excluding the two Rop fusion peptides) weresequenced, and their coding regions are shown in Table 11. Stop codonsare represented by stars, and the landmark Bgl II and EcoR I restrictionsites for the insert region are underlined. Since the ends of theoligonucleotide from which these inhibitors were constructed containedthese restriction sites, the oligonucleotide was not gel isolated whenthe libraries were prepared in order to maximize the oligonucleotideyields. Because of this, several of the inhibitory clones were found tocontain one (n−1) or two (n−2) base deletions in the randomized portionof the oligonucleotide.

TABLE 11Sequence analysis of the insert region from the top 10 inhibitoryclones and the peptides that they are predicted to encode pPep1 - 13 aaCAG GAA AGA TCT ATG GTC ACT GAA TTT TGT GGC TTG TTG GAC CAA CTG CCT TAG TAA TAG TGG AAG(SEQ ID NO: 35)                 M   V   T   E   F   C   G   L   L   D   Q   L   P   *   *   *(SEQ ID NO: 34)             341 GCT GAA ATT AAT AAG AAT TC pPep5 - 16 aaCAG GAA AGA TCT ATG TGG CGG GAC TCA TGG ATT AAG GGT AGG GAC GTG GGG TTT ATG GGT TAA AAT(SEQ ID NO: 37)                 M   W   R   D   S   W   I   K   G   R   D   V   G   F   M   G   *(SEQ ID NO: 36) AGT TTG ATA ATA AGA ATT CpPep6 - 42 aa - last 25 aa could form a hydrophobic membrane-spanning domainCAG GAA AGA TCT ATG TCA GGG GGA CAT GTG ACG AGG GAG TGC AAG TCG GCG ATG TCC AAT CGT TGG(SEQ ID NO: 39)           M S  G G  H  V T R  E   C  K S A   M  S N R W  I(SEQ ID NO: 38)ATC TAC GTA ATA AGA ATT CTC ATG TTT GAC AGC TTA TCA TCG ATA AGC TTT AAT GCG GTA GTT TAT    V I R I L  M F  D S L  S S I S  F N A  V  V Y  H S TAA CAC AGT Y *pPep7 - 6 aaCAG GAA AGA TCT ATG TAT TTG TTC ATC GGA TAA TAC TTA ATG GTC CGC TGG AGA ACT TCA GTT TAA(SEQ ID NO: 41)                  M   Y   L   F   I   G   *(SEQ ID NO: 40) TAA GAA TTC pPep8 - 21 aaCAG GAA AGA TCT ATG CTT CTA TTT GGG GGG GAC TGC GGG CAG AAA GCC GGA TAC TTT ACT GTG CTA(SEQ ID NO: 42)                 M   L   L   F   G   G   D   C   G   Q   K   A   G   Y   F   T   V   L(SEQ ID NO: 43) CCG TCA AGG TAA TAA GAA TTC  P   S   R   *   *pPep10 - 20 aa - predicted to be 45% β-sheet -amino acids 6-14CAG GAA AGA TCT ATG ATT GGG GGA TCG TTG AGC TTC GCC TGG GCA ATA GTT TGT AAT AAG AAT TCT(SEQ ID NO: 44)                 M   I   G   G   S   L   S   F   A   W   A   I   V   C   N   K   N   S(SEQ ID NO: 45) CAT GTT TGA  H   V   * pPep12 - 14 aaCAG GAA AGA TCT ATG AAC GGC CGA ACC AAA CGA ATC CGG GAC CCA CCA GCC GCC TAA ACA GCT ACC(SEQ ID NO: 47)                 M   N   G   R   T   K   R   I   R   D   P   P   A   A   *(SEQ ID NO: 46) AGC TGT GGT AAT AAG AAT TCpPep13 -18 aa - predicted to be 72% α-helical - amino acids 3-15CAG GAA AGA TCT ATG GAC CGT GAA GTG ATG TGT GCG GCA AAA CAG GAA TGG AAG GAA CGA ACG CCA(SEQ ID NO: 49)                 M   D   R   E   V   M   C   A   A   K   Q   E   W   K   E   R   T   P(SEQ ID NO: 48) TAG GCC GCG TAA TAA GAA TTC  * pPep17 - 12 aaCAG GAA AGA TCT ATG TAG CCC AAT GCA CTG GGA GCA CGC GTG TTA GGT CTA GAA GCC ACG TAC CCA(SEQ ID NO: 50)                 M   *                               M   L   G   L   E   A   T   Y   P(SEQ ID NO: 51) TTT AAT CCA TAA TAA GAA TTC  F   N   P   *   *pPep19 - 5 aaCAG GAA AGA TCT ATG AGG GGC GCC AAC TAA GGG GGG GGG AAG GTA TTT GTC CCG TGC ATA ATC TCG(SEQ ID NO: 53)                  M   R   G   A   N   * (SEQ ID NO: 52)GGT GTT GTC TAA TAA GAA TTC

Eight out of the top 10 inhibitors were predicted to encode peptidesthat terminate before the double TAA TAA termination site, which wasengineered into the oligonucleotide. Two of the inhibitors, pPep6 andpPep10, which contain deletions within the randomized portion of theoligonucleotide, are terminated beyond the EcoR I site. One of theinhibitors, pPep17, contains a termination signal just after the ATGstart codon. However, just downstream from this is a Shine Dalgamo siteand a GTG codon, which should function as the start codon.Interestingly, the start sites of several proteins such as Rop areidentical to that proposed for the pPep 17 peptide (G. Cesareni et al.,Proc. Natl. 35 Acad. Sci. USA. 79:6313-6317 (1982)). The average andmedian length for the 8 peptides whose termination signals occurredbefore or at the double TAA TAA termination site was 13 amino acids.

The characteristics of the predicted coding regions of the inhibitorpeptides proved to be quite interesting. Three out of the top 10peptides, pPep1, pPep13, and pPep17, contained a proline residue astheir last (C-terminal) amino acid. Additionally, one of the peptides,pPep12, contained 2 proline residues near the C-terminus, at the n−2 andn−3 positions. Thus there appears to be a bias for the placement ofproline residues at or near the end of several of the inhibitorypeptides. Secondary structure analysis predicted that 3 out of the 10peptides contained a known motif that could potentially form a verystable structure. pPep13, a peptide containing a C-terminal proline, ispredicted to be 72% α-helical, pPep 10 is predicted to be 45% β-sheet,and pPep6 is predicted to form a hydrophobic membrane spanning domain.

Verifying that the Inhibitory Clones do not Function as Antisense. Torule out the possibility that the bioactivity of the inhibitory clonesresulted from their functioning as antisense RNA or DNA (thushybridizing to host DNA or RNA) rather than by way of the encodedpeptides, the insert regions between the Bgl II and EcoR I sites for thetop five inhibitors from Table 10 were recloned into the pLAC11 vectorusing oligonucleotides which converted the ATG start codon to an ACGcodon thus abolishing the start site. In all five cases the newconstructs were no longer inhibitory (see Table 12), thus confirmingthat it is the encoded peptides that causes the inhibition and not theDNA or transcribed mRNA.

TABLE 12 Antisense test of the top 5 “anchorless” inhibitory peptides %inhibition % inhibition versus pLAC11 Antisense versus pLAC11 Inhibitorypeptide control construct control pPep1 26 pPep1-anti 0 pPep5 23pPep5-anti 0 pPep12 80 pPep12-anti 0 pPep13 28 pPep13-anti 0 pPep19 29pPep19-anti 0Growth rates for cells containing the induced inhibitors or antisenseconstructs were determined and then the % inhibition was calculated bycomparing these values to the growth rate of cells that contained theinduced pCyt-3 vector.Discussion

Use of the tightly regulable pLAC11 expression vector made possible theidentification of novel bioactive peptides. The bioactive peptidesidentified using the system described in this Example inhibit the growthof the host organism (E. coli) on minimal media. Moreover, bioactivepeptides thus identified are, by reason of the selection process itself,stable in the host's cellular environment. Peptides that are unstable inthe host cell, whether or not bioactive, will be degraded; those thathave short half-lives are, as a result, not part of the selectable pool.The selection system thus makes it possible to identify and characterizenovel, stable, degradation-resistant bioactive peptides in essentially asingle experiment.

The stability of the inhibitory peptides identified in this Example maybe related to the presence of certain shared structural features. Forexample, three out of the top 10 inhibitory “anchorless” (i.e., non-Ropfusion) peptides contained a proline residue as their last amino acid.According to the genetic code, a randomly generated oligonucleotide suchas the one used in this Example has only a 6% chance of encoding aproline at a given position, yet the frequency of a C-terminal prolineamong the top ten inhibitory peptides is a full 30%. This 5-fold bias infavor of a C-terminal proline is quite surprising, because although thepresence of proline in a polypeptide chain generally protectsbiologically active proteins against nonspecific enzymatic degradation,a group of enzymes exists that specifically recognize proline at or nearthe N- and C-termini of peptide substrates. Indeed, proline-specificpeptidases have been discovered that cover practically all situationswhere a proline residue might occur in a potential substrate (D. F.Cunningham et al., Biochimica et Biophysics Acta 1343:160-186 (1997)).For example, although the N-terminal sequences Xaa-Pro-Yaa- andXaa-Pro-Pro-Yaa (SEQ ID NO:54) have been identified as being protectiveagainst nonspecific N-terminal degradation, the former sequence iscleaved by aminopeptidase P (at the Xaa-Pro bond) and dipeptidylpeptidases IV and II (at the -Pro-Yaa-bond)) (Table 5, G. Vanhoof etal., FASEB J. 9:736-44 (1995); D. F. Cunningham et al., Biochimica etBiophysics Acta 1343:160-186 (1997)); and the latter sequence, presentin bradykinin, interleukin 6, factor XII and erythropoietin, is possiblycleaved by consecutive action of aminopeptidase P and dipeptidylpeptidase IV (DPPIV), or by prolyl oligopeptidase (post Pro-Pro bond)(Table 5, G. Vanhoof et al., FASEB J. 9:736-44 (1995)). Prolyloligopeptidase is also known to cleave Pro-Xaa bonds in peptides thatcontain an N-terminal acyl-Yaa-Pro-Xaa sequence (D. F. Cunningham etal., Biochimica et Biophysics Acta 1343:160-186 (1997)). Other prolinespecific peptidases acting on the N-terminus of substrates includeprolidase, proline iminopeptidase and prolinase. Prolyl carboxypeptidaseand carboxypeptidase P, on the other hand, cleave C-terminal residuesfrom peptides with proline being the preferred P₁ residue (D. F.Cunningham et al., Biochimica et Biophysics Acta 1343:160-186 (1997).

Also of interest with respect to the stability of the inhibitorypeptides, three of the top ten (30%) contained motifs that werepredicted, using standard protein structure prediction algorithms, toform stable secondary structures. One of the peptides (which also has aC-terminal proline) was predicted to be 72% α-helical. Another waspredicted to be 45% β-sheet; this peptide may dimerize in order toeffect the hydrogen bonding necessary to form the β-sheet. A third waspredicted to possess a hydrophobic membrane spanning domain. Accordingto these algorithms (see, e.g., P. Chou et al., Adv. Enzymol. 47:45-148(1978); J. Gamier et al., J. Mol. Biol. 120:97-120 (1978); P. Chou,“Prediction of protein structural classes from amino acid composition,”In Prediction of Protein Structure and the Principles of ProteinConformation (Fasman, G. D. ed.). Plenum Press, New York, N.Y. 549-586(1990); P. Klein et al., Biochim. Biophys. Acta 815:468-476 (1985)), arandomly generated oligonucleotide such as the one used in our studieswould have had no better than a 1 in a 1000 chance of generating themotifs that occurred in these peptides.

Finally, two of the three two-day inhibitors proved to be fusionpeptides in which the carboxyl terminus of the peptides was fused to theamino terminus of the Rop protein. Rop is a small 63 amino acid proteinthat consists of two antiparallel É-helices connected by a sharp hairpinloop. It is a dispensable part of the ColE1 replicon which is used byplasmids such as pBR322, and it can be deleted without causing anyill-effects on the replication, partitioning, or copy numbers ofplasmids that contain a ColE1 ori (X. Soberon, Gene. 9:287-305 (1980).Rop is known to possess a highly stable structure (W. Eberle et al.,Biochem. 29:7402-7407 (1990); S. Betz et al., Biochemistry 36:2450-2458(1997)), and thus it could be serving as a stable protein anchor forthese two peptides.

Table 13 lists naturally occurring bioactive peptides whose structureshave been determined. Most of these peptides contain ordered structures,further highlighting the importance of structural stabilization.Research on developing novel synthetic inhibitory peptides for use aspotential therapeutic agents over the last few years has shown thatpeptide stability is a major problem that must be solved if designersynthetic peptides are to become a mainstay in the pharmaceuticalindustry (J. Bai et al., Crit. Rev. Ther. Drug. 12:339-371 (1995); R.Egleton Peptides. 18:1431-1439 (1997); L. Wearley, Crit. Rev Ther DrugCarrier Syst. 8:331-394 (1991). The system described in this Examplerepresents a major advance in the art of peptide drug development bybiasing the selection process in favor of bioactive peptides thatexhibit a high degree of stability in an intracellular environment.

TABLE 13 Structural motifs observed in naturally occurring bioactivepeptides Bioactive Size in Structural Peptide Amino acids MotifReference Dermaseptin 34 α-helix Mor et al., Biochemistry, 33: 6642-6650(1994) Endorphin 30 α-helix Blanc et al., J. Biol. Chem., 258: 8277-8284(1983) Glucagons 29 α-helix Bedarkar et al., Ciba Found Symp 60: 105-121(1977) Magainins^(a) 23 α-helix Bechinger et al., Protein Sci. 2:2077-2084 (1993) Mastoparan 14 α-helix Cachia et al., Biochemistry 25:3553-3562 (1986) Melittin 26 α-helix Terwilliger et al., J. Biol. Chem.257: 6010-6015 (1982) Motilin 22 α-helix Khan et al., Biochemistry 29:5743-5751 (1990) PK1 (5-24) 20 α-helix Reed et al., Biochemistry 26:7641-7647 (1987) Secretin 27 α-helix Gronenborn, et al. FEBS Lett., 215:88-94 (1987) Atrial Natriuretic 28 disulfide bonds Misono, et al.,Biochem. Biophys. Peptide Res. Comm. 119: 524-529 (1984) Calcitonin 32disulfide bonds Barling et al., Anal. Biochem. 144: 542-552 (1985)Conotoxins^(a) 10-30 disulfide bonds Olivera, et al., J. Biol. Chem.266: 22067-22070 (1991) Defensins^(a) 29-34 disulfide bonds Lehrer, etal., Ann. Intern. Med. 109: 127-142 (1988) EETI II 29 disulfide bondsHeitz, et al., Biochemistry 28: 2392-2398 (1989) Oxytocin 9 disulfidebonds Urry, et al., Proc. Natl. Acad. Sci. USA 60: 967-974 (1968)Somatostatin 14 disulfide bonds Namboodiri, et al. J. Biol. Chem. 257:10030-10032 (1982) Vasopressin 9 disulfide bonds Fong, et al., Biochem.Biophys. Res. Comm. 14: 302-306 (1964) Bombesin 14 disordered Carmona,et al., Biochim. Biophys. Acta 1246: 128-134 (1995) Histatin 24disordered Xu, et al. J. Dent. Res. 69: 1717-1723 (1990) Substance P 11disordered Williams and Weaver, J. Biol. Chem. 265: 2505-2513 (1990)^(a)These peptides belong to multi-member families.

Example III Directed Synthesis of Stable Synthetically EngineeredInhibitor Peptides

These experiments were directed toward increasing the number ofbioactive peptides produced by the selection method described in ExampleII. In the initial experiment, randomized peptides fused to the Ropprotein, at either the N- or C-terminus, were evaluated. In the secondexperiment, nucleic acid sequences encoding peptides containing arandomized internal amino acid sequence flanked by terminal prolineswere evaluated. Other experiments included engineering into the peptidesan α-helical structural motif, and engineering in a cluster of oppositecharges at the N- and C-termini of the peptide.

Materials and Methods

Media. Rich LB and minimal M9 media used in this study was prepared asdescribed by Miller (see Example I). Ampicillin was used in rich mediaat a final concentration of 100 îg/ml and in minimal media at a finalconcentration of 50 îg/ml. IPTG was added to media at a finalconcentration of 1 mM.

Chemicals and Reagents. Extension reactions were carried out usingKlenow from New England Biolabs (Bedford, Mass.) while ligationreactions were performed using T4 DNA ligase from Life Sciences(Gaithersburg, Md.) Alkaline phosphatase (calf intestinal mucosa) fromPharmacia (Piscataway, N.J.) was used for dephosphorylation. IPTG wasobtained from Diagnostic Chemicals Limited (Oxford, Conn.).

Bacterial Strains and Plasmids. ALS225, which isMC1061/F′lacI^(q1)Z+Y+A+, was the E. coli bacterial strain used in thisstudy (see Example I). The genotype for MC1061 is araD139Δ(araABOIC-leu)7679 Δ(lac)X74 galU galK rpsL hsr−hsm+ as previouslydescribed. pLAC11 (Example I), a highly regulable expression vector, wasused to make p-Rop(C) and p(N)Rop-fusion vectors as well as the otherrandomized peptide libraries which are described below.

Construction of the p-Rop(C) Fusion Vector. The forward primer 5′TAC TATAGA TCT ATG ACC AAA CAG GAA AAA ACC GCC 3′ (SEQ ID NO:55) and thereverse primer 5′TAT ACG TAT TCA GTT GCT CAC ATG TTC TTT CCT GCG 3′ (SEQID NO:56) were used to PCR amplify a 558 bp DNA fragment using pBR322 asa template. This fragment contained a Bgl II restriction site which wasincorporated into the forward primer followed by an ATG start codon andthe Rop coding region. The fragment extended beyond the Rop stop codonthrough the Afl III restriction site in pBR322. The amplified dsDNA wasgel isolated, restricted with Bgl II and Afl III, and then ligated intothe pLAC expression vector which had been digested with the same tworestriction enzymes. The resulting p-Rop(C) fusion vector is 2623 bp insize (FIG. 7).

Construction of the p(N)Rop-Fusion Vector. The forward primer 5′AAT TCATAC TAT AGA TCT ATG ACC AAA CAG GAA AAA ACC GC 3′ (SEQ ID NO:57) and thereverse primer 5′TAT ATA ATA CAT GTC AGA ATT CGA GGT TTT CAC CGT CAT CAC3′ (SEQ ID NO:58) were used to PCR amplify a 201 bp DNA fragment usingpBR322 as a template. This fragment contained a Bgl II restriction sitewhich was incorporated into the forward primer followed by an ATG startcodon and the Rop coding region. The reverse primer placed an EcoR Irestriction site just before the Rop TGA stop codon and an Afl IIIrestriction site immediately after the Rop TGA stop codon. The amplifieddsDNA was gel isolated, restricted with Bgl II and Afl III, and thenligated into the pLAC II expression vector which had been digested withthe same two restriction enzymes. The resulting p(N)Rop-fusion vector is2262 bp in size (FIG. 8).

Generation of Rop Fusion Randomized Peptide Libraries. Peptide librarieswere constructed as described in Example II. The syntheticoligonucleotide 5′TAC TAT AGA TCT ATG (NNN)₂₀ CAT AGA TCT GCG TGC TGTGAT 3′ (SEQ ID NO:59) was used to construct the randomized peptidelibraries for use with the p-Rop(C) fusion vector, substantially asdescribed in Example II. The complementary strand of thisoligonucleotide was generated by a fill-in reaction with Klenow using anequimolar amount of the oligonucleotide primer 5′ATC ACA GCA CGC AGA TCTATG 3′ were used (SEQ ID NO:60). After extension, the resulting dsDNAwas digested with Bgl II and ligated into the pLAC11 expression vectorwhich had been digested with the same restriction enzyme andsubsequently dephosphorylated using alkaline phosphatase. Because of theway the oligonucleotide library has been engineered, either orientationof the incoming digested double-stranded DNA fragment results in afusion product.

To construct the randomized peptide libraries for use with the p(N)Ropfusion vector, the randomized oligonucleotide 5′TAC TAT GAA TTC(NNN)₂₀GAA TTC TGC CAC CAC TAC TAT 3′ (SEQ ID NO:61), and the primer 5′ ATA GTAGTG GTG GCA GAA TTC 3′ (SEQ ID NO:62) were used. After extension, theresulting dsDNA was digested with EcoRI and ligated into the pLAC11expression vector which had been digested with the same restrictionenzyme and subsequently dephosphorylated using alkaline phosphatase.Because of the way the oligonucleotide library has been engineered,either orientation of the incoming digested double-stranded DNA fragmentresults in a fusion product.

Generation of a Randomized Peptide Library Containing Terminal Prolines.Randomized amino acid peptide libraries containing two proline residuesat both the amino and the carboxy terminal ends of the peptides wereconstructed using the synthetic oligonucleotide 5′TAC TAT AGA TCT ATGCCG CCG (NNN)₁₆ CCG CCG TAA TAA GAA TTC GTA CAT 3′ (SEQ ID NO:63). Thecomplementary strand of the 93 base randomized oligonucleotide wasgenerated by filling in with Klenow using the oligonucleotide primer5′ATG TAC GAA TTC TTA TTA CGG CGG 3′ (SEQ ID NO:64). After extension,the resulting dsDNA was digested with Bgl II and EcoR I and ligated intothe pLAC11 expression vector which had been digested with the same tworestriction enzymes. Because the initiating methionine of the peptidescoded by this library is followed by a proline residue, the initiatingmethionine will be removed (F. Sherman et al, Bioessays 3:27-31 (1985)).Thus the peptide libraries encoded by this scheme are 20 amino acids inlength.

Generation of a Randomized Hydrophilic α-Helical Peptide Library. Table14 shows the genetic code highlighted to indicate certain amino acidproperties.

TABLE 14 Genetic Code Highlighted to Indicate Amino Acid Properties TTTphe h_(a) TCT ser TAT tyr b_(a) TGT cys TTC phe h_(a) TCC ser TAC tyrb_(a) TGC cys TTA leu H_(a) TCA ser TAA OCH TGA OPA TTG leu H_(a) TCGser TAG AMB TGG trp CTT leu H_(a) CCT pro B_(a) CAT his h_(a) CGT argCTC leu H_(a) CCC pro B_(a) CAC his h_(a) CGC arg CTA leu H_(a) CCA proB_(a) CAA gin h_(a) CGA arg CTG leu H_(a) CCG pro B_(a) CAG gln h_(a)CGG arg ATT ile h_(a) ACT thr AAT asn b_(a) AGT ser ATC ile h_(a) ACCthr AAC asn b_(a) AGC ser ATA ile h_(a) ACA thr AAA asn h_(a) AGA argATG met H_(a) ACG thr AAG asn h_(a) AGG arg GTT val h_(a) GCT ala H_(a)GAT asp h_(a) GGT gly B_(a) GTC val h_(a) GCC ala H_(a) GAC asp h_(a)GGC gly B_(a) GTA val h_(a) GCA ala H_(a) GAA asp h_(a) GGA gly B_(a)GTG val h_(a) GCG ala H_(a) GAG asp h_(a) GGG gly B_(a) Boldface aminoacids are hydrophobic while italicized amino acids are hydrophilic. Thepropensity for various amino acids to form α-helical structures is alsoindicated in this table using the conventions first described by Chouand Fasman (P. Chou et al., Adv. Enzymol. 47: 45-148 (1978)). H_(a) =strong α-helix former, h_(a) = α-helix former, B_(a) = strong α-helixbreaker, b_(a) = α-helix breaker. The assignments given in this tableare the consensus agreement from several different sources. Hydrophilicversus hydrophobic assignments for the amino acids were made from datafound in Wolfenden et. al. (Biochemistry. 20: 849-55 (1981)); Miller et.al. (J Mol. Biol. 196: 641-656 (1987)); and Roseman (J Mol. Biol. 200: 513-22(1988)). The propensity for amino acids to form α-helicalstructures were obtained from consensus agreements of the Chou andFasman (P. Chou et al., Adv. Enzymol. 47: 45-148 (1978); P. Chou,“Prediction of protein structural classes from amino acid compositions,”in Prediction of protein structure and the principles of proteinconformation (G. Fasman, G. D. ed.). Plenum Press, New York, N.Y.549-586 (1990)); Gamier, Osguthorpe, and Robson (J Mol. Biol. 120:97-120 (1978)); and O'Neill and DeGrado (Science. 250: 646-65 1 (1990))methods for predicting secondary structure.

By analyzing the distribution pattern of single nucleotides in thegenetic code relative to the properties of the amino acids encoded byeach nucleotide triplet, a novel synthetic approach was identified thatwould yield randomized 18 amino acid hydrophilic peptide libraries witha propensity to form α-helices. According to Table 14, the use of a[(CAG)A(TCAG)] codon mixture yields the hydrophilic amino acids His,Gln, Asn, Lys, Asp, and Glu. These amino acids are most often associatedwith α-helical motifs except for asparagine, which is classified as aweak α-helical breaker. If this codon mixture was used to build anα-helical peptide, asparagine would be expected to occur in about 17% ofthe positions, which is acceptable in an α-helical structure accordingto the secondary structure prediction rules of either Chou and Fasman(P. Chou et al., Adv. Enzymol. 47:45-148 (1978); P. Chou, “Prediction ofprotein structural classes from amino acid compositions,” in Predictionof protein structure and the principles of protein conformation (G.Fasman, G. D. ed.). Plenum Press, New York, N.Y. 549-586 (1990)) orGamier, Osguthorpe, and Robson (J. Gamier et al., J. Mol. Biol.120:97-120 (1978)). Additionally, several well-characterized proteinshave been observed to contain up to three b_(a) breaker amino acidswithin a similarly sized α-helical region of the protein (T. Creighton,“Conformational properties of polypeptide chains,” in Proteins:structures and molecular properties, W.H. Freeman and Company, N.Y.,182-186 (1993)). Since in most α-helices there are 3.6 amino acids percomplete turn, the 18 amino acid length was chosen in order to generateα-helical peptides which contained 5 complete turns. Moreover, the useof hydrophilic amino acids would be expected to yield peptides which aresoluble in the cellular cytosol.

Randomized 18 amino acid hydrophilic α-helical peptide libraries weresynthesized using the synthetic oligonucleotide 5′TAC TAT AGA TCT ATG(VAN)₁₇ TAA TAA GAA TTC TGC CAG CAC TAT 3′ (SEQ ID NO:65). Thecomplementary strand of the 90 base randomized oligonucleotide wasgenerated by filling in with Klenow using the oligonucleotide primer5′ATA GTG CTG GCA GAA TTC TTA TTA 3′ (SEQ ID NO:66). After extension theresulting dsDNA was digested with Bgl II and EcoR I and ligated into thepLAC11 expression vector which had been digested with the same tworestriction enzymes.

Generating a Randomized Peptide Library Containing the +/− Charge EndingMotif. Randomized peptide libraries stabilized by the interaction ofoppositely charge amino acids at the amino and carboxy termini weregenerated according to the scheme shown in FIG. 9. To maximize thepotential interactions of the charged amino acids, the larger acidicamino acid glutamate was paired with the smaller basic amino acidlysine, while the smaller acidic amino acid aspartate was paired withthe larger basic amino acid arginine. To construct the randomizedpeptide libraries, the synthetic oligonucleotide 5′TAC TAT AGA TCT ATGGAA GAC GAA GAC(NNN)₁₆ CGT AAA CGT AAA TAA TAA GAA TTC GTA CAT 3′ (SEQID NO:67) and the oligonucleotide primer 5′ATG TAC GAA TTC TTA TTA TTTACG TTT ACG 3′ (SEQ ID NO: 68) were used. After extension, the resultingdsDNA was digested with Bgl II and EcoR I and ligated into the pLAC11expression vector which had been digested with the same two restrictionenzymes.

For all libraries of randomized oligonucleotides, N denotes that anequimolar mixture of the four nucleotides A, C, G. and T was used, and Vdenotes that an equimolar mixture of the three nucleotides A, C and Gwas used. The resulting libraries were transformed into electrocompetentALS225 E. coli cells (Example I) under repressed conditions as describedin Example II.

Screening of Transformants to Identify Inhibitor Clones. Transformantswere initially screened using the grid-patching technique to identifyany that could not grow on minimal media as described in Example II whenthe peptides were overproduced. To verify that all the inhibitors werelegitimate, plasmid DNA was made from each inhibitory clone, transformedinto a fresh background, then checked to make sure that they were stillinhibitory on plates and that their inhibition was dependent on thepresence of the inducer, IPTG, as in Example II.

Growth Rate Analysis in Liquid Media. Inhibition strength of thepeptides was assessed by subjecting the inhibitory clones to a growthrate analysis in liquid media. Minimal or rich cultures containingeither the inhibitor to be tested or the relevant vector as a controlwere diluted to an initial OD₅₅₀ of approximately 0.01 using new mediaand induced with 1 mM IPTG. OD₅₅₀ readings were then taken hourly untilthe cultures had passed log phase. Growth rates were determined as thespectrophotometric change in OD₅₅₀ per unit time within the log phase ofgrowth, and inhibition of the growth rate was calculated for theinhibitors using the appropriate vector as a control.

Results

Isolation and Characterization of Inhibitor Peptides that are Fused atTheir Carboxy Terminal End to the Amino Terminal End of the Rop Protein.

Approximately 10,000 peptides protected by the Rop protein at theircarboxy terminal end were screened using the grid-patching techniquedescribed in Example II, and 16 two day inhibitors were isolated. Theinhibitory effects were determined as described in the Example II, usingpRop(C) as a control. Unlike the anchorless inhibitors identified inExample II that were only inhibitory on minimal media, many of the Ropfusion inhibitors were also inhibitory on rich media as well, whichreflects increased potency. As indicated in Table 15, the inhibitorsinhibited the bacterial growth rate at levels that averaged 90% inminimal media and at levels that averaged 50% in rich media. The data inTable 15 is the average of duplicate experiments.

TABLE 15 Inhibitory effects of peptide inhibitors stabilized by fusingthe carboxy terminal end of the peptide to the amino terminal end of theRop protein (Rop(C) fusion peptide inhibitors % inhibition in %inhibition in Inhibitor minimal media rich media pRop(C)1 87 47 pRop(C)299 58 pRop(C)3 85 54 pRop(C)4 98 49 pRop(C)5 95 54 pRop(C)6 99 46pRop(C)7 91 59 pRop(C)8 86 51 pRop(C)9 93 57 pRop(C)10 91 35

Isolation and Characterization of Inhibitor Peptides that are Fused atTheir Amino Terminal End to the Carboxy Terminal End of the Rop Protein.Approximately 6000 peptides protected at their amino terminal end by Ropprotein were screened using the grid-patching technique described inExample II, and 14 two day inhibitors were isolated. As observed for theRop fusion peptides isolated using the p-Rop(C) vector, most of theinhibitor peptides isolated using the p(N)Rop- vector were inhibitory onrich media as well as minimal media. The inhibitors were verified asdescribed hereinabove and subjected to growth rate analysis usingp(N)Rop- as a control in order to determine their potency. As indicatedin Table 16, the inhibitors inhibited the bacterial growth rate atlevels that averaged 90% in minimal media and at levels that averaged40% in rich media. The data in Table 16 is the average of duplicateexperiments.

TABLE 16 Inhibitory effects of peptide inhibitors stabilized by fusingthe amino terminal end of the peptide to the carboxy terminal end of theRop protein (Rop(N) fusion peptide inhibitors) % inhibition in %inhibition in Inhibitor minimal media rich media pRop(N)1 81 30 pRop(N)296 53 pRop(N)3 95 43 pRop(N)4 92 38 pRop(N)5 99 33 pRop(N)6 93 38pRop(N)7 87 34 pRop(N)8 91 44 pRop(N)9 95 37 pRop(N)10 96 40

Isolation and Characterization of Anchorless Inhibitor PeptidesContaining Two Prolines at Both Their Amino Terminal and CarboxyTerminal Ends. Approximately 7500 peptides were screened using thegrid-patching technique described in Example II, and 12 two dayinhibitors were isolated. As indicated in Table 17, the top teninhibitors inhibited the bacterial growth rate at levels that averaged50% in minimal media. The inhibitory effects were determined asdescribed in the text using pLAC11 as a control. The data in Table 17 isthe average of duplicate experiments.

TABLE 17 Inhibitory effects of peptide inhibitors stabilized by twoproline residues at both the amino and carboxy terminal ends of thepeptide % inhibition in Inhibitor minimal media pPro1 50 pPro2 49 pPro350 pPro4 59 pPro5 52 pPro6 93 pPro7 54 pPro8 42 pPro9 41 pPro10 42

Sequence analysis of the coding regions for the top ten inhibitors isshown in Table 19. The landmark Bgl II and EcoR I restriction sites forthe insert region are underlined, as are the proline residues.

Since the ends of the oligonucleotide from which these inhibitors wereconstructed contained Bgl II and EcoRI I restriction sites, theoligonucleotide was not gel isolated when the libraries were prepared inorder to maximize the oligonucleotide yields. Because of this, three ofthe inhibitory clones, pPro2, Ppro5, and pPro6 were found to containdeletions in the randomized portion of the oligonucleotide.

TABLE 18Sequence analysis of the insert region from the proline peptidespPro1 - 21 aaAGA TCT ATG CCG CCG ATT CTA TGG GGC GAA GCG AGA AAG CGC TTG TGG GGT GGG GAT CAT ACA CCG(SEQ ID NO: 69)         M   P   P   I   L   W   G   E   A   R   K   R   L   W   G   G   D   H   T   P(SEQ ID NO: 70) CCG TAA TAA GAA TTC  P   *   * pPro2 - 27 aaAGA TCT ATG CCG CCG CCG TTG GAT ATT GTG TCG GGT ATT GAG GTA GGG GGG CAT TTG TGG TGC CGC(SEQ ID NO: 71)         M   P   P   P   L   D   I   V   S   G   I   E   V   G   G   H   L   W   C   R(SEQ ID NO: 72) CGT ATT AAG AAT TCT CAT GTT TGA R   I   K   N   S   H   V   * pPro3 - 8 aaAGA TCT ATG CCG CCG GAC AAT CCG GTC CTG TGA TGA AGC GGA GGT CGA CCA AGG GGA TAT CAG CCG(SEQ ID NO: 73)          M   P   P   D   N   P  V   L   *(SEQ ID NO: 74) CCG TAA TAA GAA TTC pPro4 - 9 aaAGA TCT ATG CCG CCG CTA TTG GAC GGA GAT GAC AAA TAG ATA TAT GCG TGG TTG TTT TTC TGT CCG(SEQ ID NO: 75)          M   P   P   L   L   D   G   D   D   K  *(SEQ ID NO: 76) CCG TAA TAA GAA TTC pPro5 - 10 aaAGA TCT ATG CCG CCG AGG TGG AAG ATG TTG ATA AGA CAG TGA CAG ATG CGT TCC ATT ACT CCC GCC(SEQ ID NO: 77)          M   P   P   R   W   K   M   L   I   R   Q   *(SEQ ID NO: 78) GTA ATA AGA ATT C pPro6 - 7 aaAGA TCT ATG ATG AGA GTA GCG CCG CCG TAA TAA GAA TTC (SEQ ID NO: 79)         M   M   R   V   A   P   P   *   * (SEQ ID NO: 80) pPro7 - 14 aaAGA TCT ATG CCG CCG TTG CGC GGG GCA TGC GAT GTA TAT GGG GTA AAT TGA ATG TCT TGT GGG CCG(SEQ ID NO: 81)         M   P   P   L   R   G   A   C   D   V   Y   G   V   N   *(SEQ ID NO: 82) CCG TAA TAA GAA TTC pPro8 - 21 aaAGA TCT ATG CCG CCG GGG AGA GGG GAA GCG GTG GGA GTG ACA TGC TTG AGC GCG AAC GTG TAC CCG(SEQ ID NO: 83)         M   P   P   G   R   G   E   A   V   G   V   T   C   L   S   A   N   V   Y   P(SEQ ID NO: 84) CCG TAA TAA GAA TTC  P   *   * pPro9 - 21 aaAGA TCT ATG CCG CCG GGA AGG GTA GTG TTC TTT GTC GCT ATC TTT GTT TCC GCA ATA TGC CTC CCG(SEQ ID NO: 85)         M   P   P   G   R   V   V   F   F   V   A   I   F   V   S   A   I   C   L   P(SEQ ID NO: 86) CCG TAA TAA GAA TTC  P   *   * pPro10 - 21 aaAGA TCT ATG CCG CCG AGG TTC GCT CAT GAG AGT GTT AAA GGG CTG GGG GAC GTT ACA AAA GCT CCG(SEQ ID NO: 87)         M   P   P   R   F   A   H   E   S   V   K   G   L   G   D   V   T   K   A   P(SEQ ID NO: 88) CCG TAA TAA GAA TTC  P   *   *

All the inhibitors were found to contain two proline residues at eithertheir amino or carboxy termini as expected. Four inhibitors containedtwo proline residues at both their amino and carboxy termini, fiveinhibitors contained two proline residues at only their amino termini,and one inhibitor contained two proline residues at only its carboxyterminus.

Isolation and Characterization of Anchorless Hydrophilic InhibitorPeptides Stabilized by an α-Helical Motif. Approximately 12,000 peptideswere screened using the grid-patching technique and 5 two-day inhibitorswere isolated. The inhibitors were verified as already described for theRop-peptide fusion studies and subjected to growth rate analysis usingpLAC11 as a control in order to determine their potency. As indicated inTable 19, the inhibitor peptides inhibited the bacterial growth rate atlevels that averaged 50% in minimal media. The averaged values of twoindependent determinations are shown.

TABLE 19 Inhibitor effects of the hydrophilic α-helical peptides %inhibition in Inhibitor minimal media pHelix1 67 pHelix2 46 pHelix3 48pHelix4 45 pHelix5 42

Sequence analysis of the coding regions for the 5 inhibitors is shown inTable 20. The landmark Bgl II and EcoR I restriction sites for theinsert region are underlined. Since the ends of the oligonucleotide fromwhich these inhibitors were constructed contained these restrictionsites, the oligonucleotide was not gel isolated when the libraries wereprepared in order to maximize the oligonucleotide yields. Because ofthis, two of the inhibitory clones, pHelix2 and pHelix3, were found tocontain deletions in the randomized portion of the oligonucleotide. Thepredicted α-helical content of these peptides is indicated in Table 20according to the secondary structure prediction rules of Garnier,Osguthorpe, and Robson (J. Garnier et al., J. Mol. Biol. 120: 97-120(1978)) prediction rules.

TABLE 20Sequence analysis of the insert region from the hydrophilic α-helical peptidespHelix1 - 18 aa, 83% α-helicalAGA TCT ATG CAT GAC GAA CAA GAG GAG GAG CAC AAT AAA AAG GAT AAC GAA AAA GAA CAC TAA TAA(SEQ ID NO: 89)         M   H   D   E   Q   E   E   E   H   N   K   K   D   N   E   K   E   H   *   *(SEQ ID NO: 90) GAA TTC pHelix2 - 22 aa 68% α-helicalAGA TCT ATG CAG CAG GAG CAC GAG CAA GGC AGG ATG AGC AAG AGG ATG AAG AAT AAT AAG AAT TCT(SEQ ID NO: 91)         M   Q   Q   E   H   E   Q   G   R   M   S   K   R   M   K   N   N   K   N   S(SEQ ID NO: 92) CAT GTT TGA  H   V   * pHelix3 - 22 aa, 55% α-helicalAGA TCT ATG AAC CAT CAT AAT GAG GCC ATG ATC AAC ACA ATG AAA ACG AGG AAT AAT AAG AAT TCT(SEQ ID NO: 93)         M   N   H   H   N   E   A   M   I   N   T   M   K   T   R   N   N   K   N   S(SEQ ID NO: 94) CAT GTT TGA  H   V   * pHelix4 - 18 aa, 17% α-helicalAGA TCT ATG AAC GAC GAC AAT CAG CAA GAG GAT AAT CAT GAT CAG CAT AAG GAT AAC AAA TAA TAA(SEQ ID NO: 95)    M  N D D N Q Q E D N H D Q H K D N K * *(SEQ ID NO: 96) GAA TTC pHelix5 - 18 aa, 50% α-helicalAGA TCT ATG CAA GAG CAG GAT CAG CAT AAT GAT AAC CAT CAC GAG GAT AAA CAT AAG AAG TAA TAA(SEQ ID NO: 97)         M   Q   E   Q   D   Q   H   N   D   N   H   H   E   D   K   H   K   K   *   *(SEQ ID NO: 98) GAA TTC

According to Garnier, Osguthorpe, and Robson secondary structureprediction, all of the encoded peptides are expected to be largelyα-helical except for pHelix4. Interestingly, pHelix1, which had thehighest degree of α-helical content, was also the most potent inhibitorypeptide that was isolated in this study.

Isolation and Characterization of Anchorless Inhibitor PeptidesStabilized by an Opposite Charge Ending Motif. Approximately 20,000peptides were screened using the grid-patching technique and 6 two dayinhibitors were isolated. The inhibitors were verified as alreadydescribed for the Rop-peptide fusion studies and subjected to growthrate analysis using pLAC11 as a control in order to determine theirpotency. As indicated in Table 21, the inhibitor peptides inhibited thebacterial growth rate at levels that averaged 50% in minimal media. Theaveraged values of two independent determinations are shown.

TABLE 21 Inhibitory effects of peptide inhibitors that are stabilized bythe opposite charge ending motif % inhibition in Inhibitor minimal mediap +/− 1 41 p +/− 2 43 p +/− 3 48 p +/− 4 60 p +/− 5 54 p +/− 6 85Sequence analysis of the coding regions for the six inhibitors is shownin Table 22. The landmark Bgl II and EcoR I restriction sites for theinsert region are underlined. With the exception of p+/−4, which wasterminated prematurely, the coding regions for the inhibitors were asexpected based on the motif that was used to generate the peptidelibraries.

TABLE 22Sequence analysis of the insert region from the opposite charge ending peptidesp +/− 1 - 25 aaAGA TCT ATG GAA GAC GAA GAC GAG GGT GCG TCA GCG TGG GGA GCA GAA CTT TGG TCG TGG CAG TCG(SEQ ID NO: 99)         M   E   D   E   D   E   G   A   S   A   W   G   A   E   L   W   S   W   Q   S(SEQ ID NO: 100) GTG CGT AAA CGT AAA TAA TAA GAA TTC V   R   K   R   K   *   * p +/− 2 - 25 aaAGA TCT ATG GAA GAC GAA GAC GGT CTA GGC ATG GGG GGT GGG TTG GTC AGG CTC ACT TTA TTA TTC(SEQ ID NO: 101)         M   E   D   E   D   G   L   G   M   G   G   G   L   V   R   L   T   L   L   F(SEQ ID NO: 102) TTC CGT AAA CGT AAA TAA TAA GAA TTC F   R   K   R   K   *   * p +/− 3 - 25 aaAGA TCT ATG GAA GAC GAA GAC GGG GAG AGG ATC CAG GGG GCC CGC TGT CCA GTA GCG CTG GTA GAT(SEQ ID NO: 103)         M   E   D   E   D   G   E   R   I   Q   G   A   R   C   P   V   A   L   V   D(SEQ ID NO: 104) AGA CGT AAA CGT AAA TAA TAA GAA TTC R   R   K   R   K   *   * p +/− 4 - 11 aaAGA TCT ATG GAA GAC GAA GAC GAC AGG GGG CGT GGG GGG TAG CTT TAA GTT GCG CTA AGT TGC GAG(SEQ ID NO: 106)          M   E   D   E   D   D   R   G   R   G   R   *(SEQ ID NO: 105) ATA CGT AAA CGT AAA TAA TAA GAA TTC p +/− 5 -25 aaAGA TCT ATG GAA GAC GAA GAC GGG GGG GCC GGG AGG AGG GCC TGT CTT TGT TCC GCG CTT GTT GGG(SEQ ID NO: 107)         M   E   D   E   D   G   G   A   G   R   R   A   C   L   C   S   A   L   V   G(SEQ ID NO: 108) GAA CGT AAA CGT AAA TAA TAA GAA TTC E   R   K   R   K   *   * p +/−6 - 25 aaAGA TCT ATG GAA GAC GAA GAC AAG CGT CGC GAG AGG AGT GCA AAA GGG CGT CAT GTC GGT CGG TCG(SEQ ID NO: 109)         M   E   D   E   D   K   R   R   E   R   S   A   K   G   R   H   V   G   R   S(SEQ ID NO: 110) ATG CGT AAA CGT AAA TAA GAC TGT  M   R   K   R   K   *Discussion

In Example II, where fully randomized peptides were screened forinhibitory effect, only three peptides (one “anchorless” and twounanticipated Rop fusions resulting from deletion) were identified outof 20,000 potential candidates as a potent (i.e., two day) inhibitor ofE. coli bacteria. Using a biased synthesis as in this Example, it waspossible to significantly increase the frequency of isolating potentgrowth inhibitors (see Table 23).

TABLE 23 Summary of the frequency at which the different types ofinhibitor peptides can be isolated Frequency at which a two dayinhibitor peptide can Type of inhibitor peptide be isolated Referenceanchorless 1 in 20,000 Example II protected at the C-terminal 1 in 625This example end via Rop protected at the N-terminal 1 in 429 Thisexample end via Rop protected at both the C- 1 in 625 This exampleterminal and N-terminal end via two prolines protected with an α-helix 1in 2,400 This example structural motif protected with an opposite 1 in3,333 This example charge ending motif

Many more aminopeptidases have been identified than carboxypeptidases inboth prokaryotic and eukaryotic cells (J. Bai, et al., Pharm. Res. 9:969-978 (1992); J. Brownlees et al., J. Neurochem. 60:793-803 (1993); C.Miller, In Escherichia coli and Salmonella typhimurium cellular andmolecular biology, 2nd edition (Neidhardt, F. C. ed.), ASM Press,Washington, D.C. 1:938-954 (1996)). In the Rop fusion studies, it mighthave therefore been expected that stabilizing the amino terminal end ofthe peptide would have been more effective at preventing the action ofexopeptidases than stabilizing the carboxy end of the peptides.Surprisingly, it was found that stabilizing either end of the peptidecaused about the same effect.

Peptides could also be stabilized by the addition of two prolineresidues at the amino and/or carboxy termini, the incorporation pfopposite charge ending amino acids at the amino and carboxy termini, orthe use of helix-generating hydrophilic amino acids. As shown in Table23, the frequency at which potent inhibitor peptides could be isolatedincreased significantly over that of the anchorless peptidescharacterized in Example II.

These findings can be directly implemented to design more effectivepeptide drugs that are resistant to degradation by peptidases. In thisexample, several strategies were shown to stabilize peptides in abacterial host. Because the aminopeptidases and carboxypeptidases thathave been characterized in prokaryotic and eukaryotic systems appear tofunction quite similarly (C. Miller, In Escherichia coli and Salmonellatyphimurium cellular and molecular biology, 2nd edition (Neidhardt, F.C. ed.), ASM Press, Washington, D.C. 1:938-954 (1996); N. Rawlings etal., Biochem J. 290: 205-218 (1993)), the incorporation of on or more ofthese motifs into new or known peptide drugs should slow or prevent theaction of exopeptidases in a eukaryotic host cell as well.

Example IV Confirmation of the Stabilizing Effects of Proline Residuesusing an In Vitro System

To extend the in vivo studies described above, an in vitro system fordirectly assessing peptide stability was developed. In the in vitrosystem, peptides to be tested were mixed with a cellular extractcontaining the proteases and peptidases present in a particular celltype. To validate this approach, the stability or half-life of arandomized biotinylated peptide initially was measured using bothwild-type bacterial extracts and bacterial extracts that were deficientin known proteases or peptidases.

Material and Methods

Bacterial strains. The bacterial strains used in this study are shown inTable 24. MG1655 clpP::cam was constructed by transducing MG1655 tochloramphenicol resistance using a P1 lysate that was prepared fromSG22098.

TABLE 24 Bacterial strains Strain Genotype Reference E. coli strainsMG1655 F-λ- Guyer, M. S. et al., 1980* MG1655 F-λ-lon::Tn10 Carol Gross,University of lon::Tn10 California, San Francisco MG1655 F-λ-clpP::camThis study clpP::cam SG22098 F-λ-araD139 Δ(lac)U169 rpsL150 thi MichaelMaurizi, National f1bB5301 deoC7 ptsF25 clpP::cam Cancer Institute S.typhimurium LT2 strains TN1379 leuBCD485 Charles Miller, University ofIllinois TN1727 leuBCD485 pepA16 pepB11 pepN90 pepP1 Charles Miller,University of pepQ1 pepT1 ΔsupQ302(proAB pepD) optA1 Illinoiszxx848::Tn5 dcp-1 zxx845::Tn10 *Guyer, M. S. et al., Cold Spring HarborSymp. Quant. Biol. 45: 135-140 (1980).

Media. Bacterial cells were grown in LB media; yeast cells were grown in1.0% yeast extract, 2.0% peptone, 2.0% glucose; human HeLa cells (ATCCCCL-2) and colon CCD-18Co cells (ATCC CRL-1459) were grown in MinimalEssential Medium Eagle (ATCC 30-2003) with Earle's balanced saltsolution, 0.1 mM non-essential amino acids, 2.0 mM L-glutamine, 1.0 mMsodium pyruvate, 1.5 g/L NaHCO₃, and 10% fetal bovine serum; and humansmall intestine FHs74 Int cells (ATCC CCL-241) were grown in Hybri-Caremedia (ATCC 46-X) with 1.5 g/L NaHCO₃ and 10% fetal bovine serum.

Preparation of the extracts. For bacteria and yeast, 500 mL of cellswere grown to an OD₅₅₀ of 0.5, centrifuged, washed twice with T₁₀E_(0.1)(10.0 mM Tris; pH 8.0, 0.1 mM EDTA; pH 8.0) and resuspended in 2.0 mL of10.0 mM Tris; pH 8.0. For human cells, 10-50 75 cm² T flasks were seededand allowed to grow to 95% confluency in a 37° C. incubator with 5% CO₂atmosphere. Each flask was then washed with HBSS (0.4 g/L KCl, 0.06 g/LKH₂PO₄, 8 g/L NaCl, 0.35 g/L NaHCO₃, 0.048 g/L Na₂HPO₄, 1.0 g/L Glucose)that contained 0.125 mM EDTA; pH 8.0. To liberate the cells, the flaskswere treated with 1.5 mL of HBSS that contained 0.25% trypsin and 0.5 mMEDTA; pH 8.0. The trypsin was neutralized by adding 5 mL of media with10% fetal bovine serum to each flask. The cells were centrifuged, washedwith HBSS that contained 0.125 mM EDTA; pH 8.0, washed twice with HBSSlacking glucose and ETDA, and resuspended in 2.0 mL of 10.0 mM Tris; pH8.0. All cell suspensions were lysed with three passes at 15,000 psi ina French Pressure cell maintained at 4° C. The lysates were thencentrifuged at 15,000 rpm, 4° C., for 10 minutes to pellet debris andunlysed cells and the supernatant was saved as the cell extract. Toprepare rat serum, one 300 g Sprague Dawley rat was euthanized with CO₂and a heart puncture was performed to draw the blood which wasimmediately transferred to a tube and centrifuged at 4° C., 10,000 rpm,for 10 minutes. The cleared serum was removed with a pipette except for1 cm of serum at the interface with the blood cell pellet.

Peptide synthesis. The following randomized biotinylated peptides weresynthesized by Sigma Genosys (The Woodlands, Texas):

Unprotected XXXXXX[KBtn]XXXXXA P at both ends PXXXX[KBtn]XXXXP PP atboth ends PPXXXX[KBtn]XXXXPP APP at both ends APPXXXX[KBtn]XXXXPPA APPamino APPXXXX[K-Btn]XXXXA APP carboxyl AXXXX[K-Btn]XXXXPPA Acetylated(Ac)AXXXXX[KBtn]XXXXXA Amidated XXXXXX[KBtn]XXXXXA(NH₂) CyclizedCXXXXXX[KBtn]XXXXXXCwhere A denotes the L-amino acid alanine, P denotes the L-amino acidproline, X denotes an equimolar mixture of the 20 natural L-amino acidsexcept for proline, and KBtn denotes the L-amino acid lysine to whichbiotin has been attached.

To ensure that the length of the randomized portion of the peptides didnot affect the degradation profiles, we also tested the unprotectedpeptides XXXx[KBtn]XXXXA and AXXXX[KBtn]XXXXA. Their half-lives weredetermined to be within 5% of the XXXXXX[KBtn]XXXXXA peptide which wasused as the control for these studies.

In vitro degradation assay. All extracts were used at a finalconcentration of 10 mg/mL, except for the S. typhimurium extracts, whichwere used at a final concentration of 25 mg/mL. The cell extract (50 μL)was mixed with 50 μL of a peptide at a concentration of 1 mg/mL in 10 mMTris; pH 8.0 and incubated at 37° C. Aliquots were removed (10 μL) at30, 60, 90, or 120 minute intervals, placed into 90 μL of SDS-PAGEgradient gel buffer, boiled for 5 minutes, and electrophoresed through a10-20% tricine gradient gel. The gel was blotted onto a nitrocellulosemembrane and the resulting Western blot was treated with NeutrAvidinHorseradish Peroxidase Conjugate and SuperSignal West Dura ExtendedDuration Chemiluminescent Substrate (Pierce, Rockford, Ill.). Thebiotinylated peptides were then visualized by exposing the blots toautoradiography film and the resulting bands were quantified using theAlphaEase 5.5 Densitometry Program from Alpha Innotech, San Leandro,Calif.

Results

The proteases and peptidases have been well characterized in E. coli andS. typhimurium. In E. coli, the two main proteases that have been shownto have a role in peptide degradation are Lon and ClpP, which areencoded respectively by the lon and clpP genes. In S. typhimurium,numerous peptidases have been identified, and strains have beenconstructed that delete several of the peptidases. Using extractsprepared from E. coli strains that contained lon or clpP deletions and aS. typhimurium strain in which nine peptidase genes were deleted,half-lives were determined for the unprotected randomized biotinylatedcontrol peptide. As shown in Table 25, deletion of the Lon proteasecaused the peptide's half-life to increase by 6.5 fold, deletion of theClpP protease caused the peptide's half-life to increase by 1.8 fold,and deletion of multiple peptidases caused the peptide's half-life toincrease by 7.1 fold. These results prove that the in vitro systemprovides an accurate method by which to assess peptide stability.

TABLE 25 Peptide degradation in protease and peptidase deficientextracts. Strain from which extract was prepared* Peptide half-life inminutes MG1655 44.9 MG1655 lon::Tn10 290.6 MG1655 clpP::cam 82.5 TN137942.0 TN1379 dcp-1 optA1 pepA16 298.5 pepB11 ΔpepD pepN90 pepP1 pepQ1pepT1 *Because of the decreased potency of S. typhimurium extractsrelative to E. coli extracts, the S. typhimurium extracts were used at aconcentration of 25 mg/mL.

With the system validated, the stabilizing effects of proline residueswere analyzed. Three randomized biotinylated peptides were tested usingextracts prepared from bacterial (wild-type E. coli), Baker's yeast(wild-type Saccharomyces cerevisiae), human (HeLa) cells, humanintestine and colon cells, and rat serum. One randomized peptide wasunprotected, while the other two peptides were stabilized on both the N-and C-termini with a Pro (P) motif, a Pro-Pro motif (PP), or anAla-Pro-Pro motif (APP). The results are shown in the Table 26.

TABLE 26 The effect of proline-containing stabilizing groups on peptidedegradation Peptide half-lives in minutes Peptide Peptide Peptideprotected at protected protected at Unprotected both ends at both bothends by Extract peptide by P ends by PP APP E. coli 44.9 38.2 51.1 69.8S. cereviseae 23.3 44.4 99.0 156.0 Human HeLa 90.8 ND 423.4 1,054.3Human 121.6 99.2 166.3 171.8 Intestine Human 58.1 64.5 76.1 109.2 ColonRat serum 54.1 80.7 85.3 154.5 ND: not determined

As the data indicate, the APP motif offered significantly moreprotection than the PP motif, which provided better protection than theP motif.

Table 27 shows the results of degradation studies on peptides thatcontain the APP motif at either or both of the amino or carboxyltermini.

TABLE 27 The effect of APP stabilizing groups on peptide degradationPeptide half-lives in minutes APP APP at both APP Amino Carboxyl ExtractUnprotected ends terminus terminus E. coli 44.9 69.8 99.6 54.6 S.cereviseae 23.3 156.0 86.0 44.4 Human Intestine 121.6 171.8 200.7 99.0Human Colon 58.1 109.2 144.0 95.1 Rat serum 54.1 154.5 165.3 121.2

The data show that APP at only the amino terminus offers slightly betterprotection than APP at both termini, and that APP at only the aminoterminus offers significantly better protection than APP at only thecarboxyl terminus.

Table 28 shows the results of degradation studies on peptides thatcontain the APP motif at the N- or C-terminus compared to peptides thatare acetylated at their amino terminus, amidated at their carboxylterminus, or cyclized.

TABLE 28 The effect of APP stabilizing groups on peptide degradation incomparison to acetylation, amidation or cyclization. Peptide half-livesin minutes Extract Unprotected APP Amino Acetylated APP CarboxylAmidated Cyclized E. coli 44.9 99.6 34.9 54.6 46.7 52.3 S. cereviseae23.3 86.0 44.2 44.4 73.9 145.0 Rat serum 54.1 165.3 67.3 121.2 75.7217.2The data clearly shows that APP at the amino terminus offers betterprotection than amidating the carboxyl terminus or acetylating the aminoterminus, and is almost as good as cyclization.

Example V Bioactivity of Natural Galanin, APP-Galanin, andAPP-Galanin-PPA

Radio immunoassays (RIAs) were performed to determine the ability ofgalanin and its APP derivatives to displace radiolabeled galanin fromits receptor. Binding (displacement) constants were then calculated fromthis data.

Natural galanin Ki = 5.21 × 10⁻⁹ APP-galanin Ki = 6.42 × 10⁻⁹APP-galanin-PPA Ki = 9.46 × 10⁻⁹

As the data shows the binding constants for the APP derivatives were inthe same range as natural galanin and thus these compounds were able tointeract with the galanin receptor in a manner similar to naturalgalanin.

Example VI In vivo Glucagon, APP-Glucagon and APP-Glucagon-PPADegradation

A catheter was placed in the right jugular vein of six MaleSprague-Dawley rats for dosing and sampling. Two rats were used for eachof the three compounds that were tested. The rats received anintravenous bolus injection of the peptide, and serial blood samples(0.3 ml) were obtained. The glucagon was extracted from plasma byorganic protein precipitation and quantified by electrospray LC-MS.

The presence of the APP motif affected both the half-life of glucagon aswell as the rate at which it is cleared from the body. The data (Table29) suggests that a significant portion of the glucagon harboring theAPP motif becomes sequestered and thus is much more resistant todegradation. It should be noted that significantly more APP-glucagon-PPAand APP-glucagon is present at 20 and 60 minutes than would be predicteddue to its half-life.

TABLE 29 Half-life Percent remaining Percent remaining Peptide inminutes after 20 minutes after 60 minutes Glucagon 1.031 0.2 0.0APP-Glucagon-PPA 1.555 3.0 ND APP-Glucagon 2.253 7.3 8.6 ND (notdetermined)

Sequence Listing Free Text

-   SEQ ID NO:2-   peptide sequence having opposite charge ending motif-   SEQ ID NOs:3, 4-   stabilized angiotensin-   SEQ ID NOs:6-19, 24-28, 55-58, 60, 62, 64, 66, 68-   primer-   SEQ ID NOs:20-22-   primer fragment-   SEQ ID NOs:23, 59, 61, 63, 65, 67-   randomized oligonucleotide-   SEQ ID NOs:29-33-   antisense oligonucleotide-   SEQ ID NOs:34, 36, 39, 40, 43, 45, 46, 48, 51, 52, 70, 72, 74, 76,    78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 105, 108,    110-   stabilized peptide-   SEQ ID NOs:35, 37, 38, 41, 42, 44, 47, 49, 50, 53, 69, 71, 73, 75,    77, 79, 81, 83, 85, 87, 89, 91, 93, 95, 97, 99, 101, 103, 106, 107,    109 nucleic acid encoding stabilized peptide-   SEQ ID NO:54-   N-terminal protective sequence-   SEQ ID NO:111-115-   α-helical moieties

The foregoing detailed description and examples have been given forclarity of understanding only. No unnecessary limitations are to beunderstood therefrom. The invention is not limited to the exact detailsshown and described, for variations obvious to one skilled in the artwill be included within the invention defined by the claim.

1. An isolated nucleic acid encoding a stabilized polypeptide, wherein said stabilized polypeptide comprises a bioactive peptide and a first stabilizing group covalently linked to a terminus of said bioactive peptide, and a second stabilizing group covalently linked to the other terminus of said bioactive peptide, wherein said first stabilizing group is heterologous to the bioactive peptide and lacks the capacity to participate in the formation of an intramolecular disulfide bond within the polypeptide, and wherein said second stabilizing group is Xaa_(n)-Pro-, Xaa_(n)-Pro- Pro-, Pro-Xaa_(n) or -Pro-Pro-Xaa_(n), wherein Xaa is any amino acid and n=1 or
 2. 2. The isolated nucleic acid of claim 1, wherein at least one Xaa is Ala.
 3. A method of making a stabilized polypeptide, said method comprising: a) providing host cells transformed with the nucleic acid of claim 1; b) expressing said stabilized polypeptide encoded by said nucleic acid; and c) recovering said stabilized polypeptide.
 4. The method of claim 3, wherein said host cells are bacteria.
 5. The method of claim 3, wherein said host cells are eukaryotic host cells.
 6. The method of claim 3, wherein the second stabilizing group is heterologous to the bioactive peptide.
 7. The method of claim 6, wherein said first and said second heterologous stabilizing groups are the same.
 8. A method of making a polypeptide comprising: providing a bacteriophage that comprises the nucleic acid of claim 1; and culturing said bacteriophage under conditions to cause the bacteriophage to express the polypeptide encoded by said nucleic acid and display it on the surface of the bacteriophage.
 9. The method of claim 8, wherein the stabilizing group is covalently linked to the N-terminus of the bioactive peptide.
 10. The method of claim 8, wherein the stabilizing group is covalently linked to the C-terminus of the bioactive peptide.
 11. The method of claim 8 further comprising cleaving the polypeptide from the host cell surface to yield a stabilized bioactive peptide comprising the bioactive peptide and the stabilizing group.
 12. A vector comprising an expression control sequence operably linked to a nucleic acid sequence encoding a stabilized polypeptide, wherein said stabilized polypeptide comprises a bioactive peptide and a first stabilizing group covalently linked to one of said bioactive peptide's termini, wherein said first stabilizing group is Xaa_(n)-Pro-, Xaa_(n)-Pro-Pro-, -Pro-Xaa_(n) or -Pro-Pro-Xaa_(n), wherein Xaa is any amino acid and wherein n=1 or
 2. 13. The vector of claim 12, wherein said stabilized polypeptide further comprises a second stabilizing group covalently linked to the other terminus of said bioactive peptide, wherein the second stabilizing group is heterologous to the bioactive peptide.
 14. The vector of claim 12, wherein said vector further comprises a tightly regulable expression control sequence operably linked to said nucleic acid sequence encoding said stabilized polypeptide.
 15. The vector of claim 14, wherein said tightly regulable expression control sequence is from a wild-type E. coli lac promoter/operator region.
 16. The vector of claim 14, wherein said expression control sequence comprises the auxiliary operator O3, the CAP binding region, the −35 promoter site, the −10 promoter site, the operator O1, lacZ Shine-Dalgarno sequence, and a spacer sequence between the end of the Shine-Dalgarno sequence and the start codon of said nucleic acid sequence.
 17. The vector of claim 16, wherein said spacer region is 5 to 10 nucleotides in length.
 18. The vector of claim 12, wherein said vector is pLAC11 having ATCC Accession No.
 207108. 19. An isolated nucleic acid encoding a stabilized polypeptide, wherein said stabilized polypeptide comprises a bioactive peptide and a stabilizing group covalently lined to one or both of said bioactive peptide's termini, wherein said stabilizing group is Xaa_(n)-Pro-Pro-, or -Pro-Pro-Xaa_(n), wherein Xaa is any amino acid and n=1 or
 2. 20. The nucleic acid of claim 19 wherein a stabilizing group is covalently linked to each of said bioactive peptide's N-terminus and C-terminus.
 21. The nucleic acid of claim 20 wherein the stabilizing group covalently lined to the N-terminus and the stabilizing group covalently linked to the C-terminus of said bioactive peptide are heterologous.
 22. The nucleic acid of claim 19 wherein said bioactive peptide is selected from the group consisting of insulin, glucagon, calcitonin, somatostatin, gonadotrophin and secretin. 